Novel-bis-benzylidine piperidone proteasome inhibitor with anticancer activity

ABSTRACT

We describe a bis-benzylidine piperidone, RA190, which covalently binds to the ubiquitin receptor RPN13 (ADRM1) in the 19S regulatory particle and inhibits proteasome function, triggering rapid accumulation of polyubiquitinated proteins. Multiple myeloma lines, even those resistant to bortezomib, were sensitive to RA190 via ER stress-related apoptosis. RA190 stabilized targets of human papillomavirus (HPV) E6 oncoprotein, and preferentially killed HPV-transformed cells. After p.o. or i.p. dosing of mice, RA190 distributed to plasma and major organs excepting brain, and potently inhibited proteasome function in skin and muscle. RA190 administration i.p. profoundly reduced growth of multiple myeloma and ovarian cancer xenografts, and oral RA190 treatment retarded HPV+ syngeneic mouse tumor growth, without impacting spontaneous HPV-specific CD8+ T cell responses, suggesting its therapeutic potential. The bis-benzylidine piperidone RA190 is a new orally-available proteasome inhibitor. Multiple myeloma, cervical and ovarian cancers are particularly sensitive to RA190.

CROSS-REFERENCE TO PRIOR APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.14/889,768, now U.S. Pat. No. 9,913,834, filed Nov. 6, 2015, which is aU.S. National Stage Application of International Application No.PCT/US2014/037031, filed May 6, 2014, which claims the benefit andpriority of U.S. Provisional Patent Applications Ser. Nos. 61/820,884(filed 8 May 2013) and 61/838,156 (filed 21 Jun. 2013) whichapplications are incorporated herein by reference to the extentpermitted by applicable statute and regulation.

U.S. GOVERNMENT SUPPORT

This invention was made with government support under grant numberCA098252, awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION Area of the Art

The present invention relates to a class of novel molecules with Michaelacceptors. These molecules are based from a bis-benzylidine piperidonebackbone and can be used as therapeutic agents against various types ofcancers. Specifically, these molecules work as proteasome inhibitors andbind to the RPN13 subunit of the 19S regulatory particle.

Description of the Background Art

Protein degradation is exquisitely regulated within the cell to maintainprotein homeostasis and eliminate misfolded or damaged proteins.³²Targeted degradation of regulatory proteins by the ubiquitin-proteasomesystem (UPS) is central to many signaling cascades including those thatgovern cell proliferation and is exploited by many infectious agents.¹²The degradation of a target protein is signaled by repeated covalentlinkage of ubiquitin mediated by E3 ubiquitin ligases, of which hundredshave been described. Upon the attachment to the target of extendedchains of ubiquitin, each conjugated via lysine 48, thepoly-ubiquitinated proteins are recognized by two proteasome subunits,RPN10 and RPN13 within the 19S regulatory particle (RP).^(19,30,31) The19S RP recycles the ubiquitin by its removal from the target protein,and unfolds the target protein while passing it to the 20S core particleof the proteasome for degradation. RPN13 binds to both UCH37, enhancingits deubiquitinase activity²⁸, and to RPN2 that modulates thetranslocation and subsequent degradation of substrates by the 20S.¹¹ The20S core particle contains three catalytic subunits, β1, β2 and β5, withcaspase, trypsin, and chymotrypsin-like activities respectively.³²Degradation occurs progressively via nucleophilic attack of thesubstrate amide bond by a Threonine within the β-subunit active site.The inhibitors bortezomib and carfilzomib principally inhibitchymotrypsin-like proteolysis.⁷

The increased reliance upon proteasomal function in cancer cells canprovide a therapeutic window.¹⁷ Bortezomib was approved for thetreatment of relapsed multiple myeloma (MM) and mantle cell lymphoma⁷,and Carfilzomib was recently approved for patients with MM progressionwhile on or after treatment with bortezomib and an immunomodulatoryagent. The efficacy of these proteasome inhibitors has been attributedto the activation of the unfolded protein response (UPR) and endoplasmicreticulum stress due to toxic accumulation of protein aggregates,inhibition of NF-κB and TNFα signaling, increases in reactive oxygenspecies (ROS) and stabilization of tumor suppressors such as p53.¹⁰ Inhuman papillomavirus (HPV)-related cancers the E6 viral oncoproteindrives transformation by co-opting the cellular E3 ubiquitin ligase E6APto polyubiquitinate target E6-binding proteins, notably tumorsuppressors such as p53 and PDZ-family members including DLG-1, andtrigger their rapid degradation.^(18,25,26) Preclinical findings suggestthat HPV-transformed cells are preferentially sensitive to bortezomib asit recovers their levels of E6-targetted tumor suppressor proteins, andtriggers apoptosis.^(23,32)

Unfortunately, bortezomib induces thrombocytopenia and neuropathy(associated with off-target activity,⁴ and the emergence of diseaseresistance remains a clinically significant problem.²⁹ Carfilzomib hassimilar issues. New orally delivered drugs targeting distinct activitiesof the proteasome are needed to increase dosing flexibility, overcomeresistance and reduce side effects. ⁹ Here we describe a new compound,RA190, which is orally bioavailable, which inhibits proteasomaldegradation by binding to a novel proteasome target, the ubiquitinreceptor RPN13, which and shows promising activity againstbortezomib-resistant MM, ovarian and HPV-associated cancers.

SUMMARY OF THE INVENTION

The present invention relates to a class of novel molecules containingMichael acceptors. In some embodiments, these molecules can be basedfrom a bis-benzylidine piperidone structure and can also be used astherapeutic agents against various types of cancers. The moleculesdescribed herein can bind to RPN13 cysteine 88 in the ubiquitin- andproteasome-binding Pru domain, rather than the UCH37-interaction domain,and inhibit proteosomal function. Furthermore, residues surrounding C88can interact with the molecules of the invention. Molecules of theinvention can bind sub-stoichiometrically to proteasome²⁷ and exist freeof proteasome in cells. The molecules of this invention are potentproteasome inhibitors of a variety of cancer cells, especially thosethat are HPV⁺ and/or those expressing high rates of protein synthesissuch as ovarian and colon cancer and multiple myeloma.

In addition, the molecules of the invention comprise an enone moiety.Elimination of Michael acceptor properties by the addition of thiol orcomplete removal of the enone moiety nullify drug activity in cytotoxicand functional assays. In addition, substitution of L- forD-phenylalanine or conversion of the carboxyl moiety to oxime reducesthe potency of the molecules.

In some embodiments, the molecules of the invention can be formulated in20% (w/v) b-hydroxyisopropyl-cyclodextrin in water. In such cases, theoral availability was only approximately 7% that of the i.p. deliverybut proved sufficient for significant antitumor effects and proteasomalinhibition in vivo. The molecules are also effective via topicaladministration at 4% in Cremophor, suggesting potential for treatment ofHPV+ intraepithelial neoplasia for cancer prevention.

Another important feature of the molecules of the invention is their lowtoxicity. The side effects of bortezomib and carfilzomib, includingneuropathy and thrombocytopenia, remain important clinical concerns. ²⁹RPN13 is one of two major ubiquitin receptors in the RP,^(14,19,30,31)and Rpn13 knockout (KO) mice are viable, suggesting that moleculestargeting RPN13 (such as those described in this invention) may have afavorable toxicity profile.² Indeed, oral treatment with at least onebis-benzylidine piperidone molecule was well tolerated, producing nosignificant difference in hematologic or clinical chemistry parametersas compared with vehicle, and it did not affect weight gain orcompromise spontaneous antitumor immunity, further suggesting apromising safety profile. Additionally, unlike many other anti-cancerchemotherapeutic agents, the molecules of the invention described hereindo not compromise immune function. Specifically, they do not compromiseE7-specific CD8+ T cell response to TC-1 tumors thus implying that itmay therefore be possible to combine treatment of cervical cancer withat least one molecule described herein (RA190) and therapeutic vaccinestargeting HPV E6 and/or E7.³⁶

In addition, in some embodiments, the molecules of the invention can beused synergistically with current cancer therapeutics that also targetproteasome function (such as bortezomib). This can especially be thecase in instances where the second therapeutic targets a distinctcomponent of the proteasome and/or resistance to the second therapeutichas occurred.

DESCRIPTION OF THE FIGURES

FIG. 1A is a graph showing percent cell viability;

FIG. 1B is a graph showing percent cell viability;

FIG. 1C is a graph showing percent cell viability;

FIG. 1D is a graph showing percent cell viability;

FIG. 1E is a graph showing percent cell viability;

FIG. 1F is a graph showing percent cell viability;

FIG. 1G is a graph showing percent cell viability;

FIG. 1H is a graph showing percent cell viability;

FIG. 1I is a western blot of lysates from HeLa cells treated with RA190(190), RA190ME (190ME), or bortezomib (Bz) for 4 hr (left) or 12 hr(right) at the concentrations indicated;

FIG. 1J shows luciferase activity of HeLa cells transiently transfectedwith either tetra-ubiquitin-fused firefly luciferase (4UbFL) or FLplasmids as a function of proteasome inhibition by various compounds.

FIG. 2A is an image of a sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) assay;

FIG. 2B is an image of an SDS-PAGE assay;

FIG. 2C is an image of an SDS-PAGE assay;

FIG. 2D is an image of an SDS-PAGE assay;

FIG. 3A is an image of a heteronuclear single quantum correlation (HSQC)experiment showing how RA190 interacts with RPN13;

FIG. 3B is an image of a heteronuclear single quantum correlation (HSQC)experiment showing how RA190 interacts with RPN13;

FIG. 3C is a graph of a Liquid Chromatography-mass spectrometry (LC-MS)experiment as indicated;

FIG. 3D is a graph of a Liquid Chromatography-mass spectrometry (LC-MS)experiment as indicated;

FIG. 3E is a graph of a Liquid Chromatography-mass spectrometry (LC-MS)experiment as indicated;

FIG. 3F is a graph of a Liquid Chromatography-mass spectrometry (LC-MS)experiment as indicated;

FIG. 3G is a graph showing HSQC spectra of ¹⁵N-labeled RPN13 PruC^(60,80,121) A and after RA190 incubation;

FIG. 4A is a bar graph depicting the amino acid residues implicated inthe RA190 and RPN13 interaction;

FIG. 4B is a bar graph depicting the amino acid residues implicated inthe RA190 and RPN13 interaction;

FIG. 4C is a model showing the lowest energy modeled structure for humanRPN13 Pru˜RA190;

FIG. 5A shows an immunoblot analysis for ATF-4 and actin in MM.1S cellseither untreated (C), or treated with RA190 (190) or bortezomib (Bz) forthe indicated times;

FIG. 5B shows an immunoblot analysis for ATF-4 in HeLa cells eitheruntreated (C), or treated with 1 μM of RA190 (190) or 1 μM bortezomib(Bz) for 6 hr;

FIG. 5C is a graph showing mRNA levels of CHOP-10 expression;

FIG. 5D is a graph showing mRNA levels of CHOP-10 expression;

FIG. 5E is a graph showing mRNA levels of XBP1 expression;

FIG. 5G is an immunoblot analysis for p53 and β-tubulin in the indicatedcell line either untreated (C) or treated with 1 μM RA190 (190) orBortezomib (Bz) for 24 hr (top panel) or for the indicated times (bottompanel);

FIG. 5H is an immunoblot assays for p21, Puma, Bax, Bak, and hDLG-1 atthe time points indicated;

FIG. 6A is a graph showing Annexin-V expression in untreated cells;

FIG. 6B is a graph showing Annexin-V expression in cells treated withRA190;

FIG. 6C is a graph showing Annexin-V expression in cells treated withRA190ME;

FIG. 6D is a graph showing Annexin-V expression in cells treated withBz;

FIG. 6E is a graph showing Annexin-V expression in untreated cells;

FIG. 6F is a graph showing Annexin-V expression in cells treated withRA190;

FIG. 6G is a graph showing Annexin-V expression in cells treated withBz;

FIG. 6H is a bar graph showing the amount of active caspase-3 in cells;

FIG. 6I is a representative flow cytometry analysis of MM.1S cellstreated as in (FIGS. 6E-6G) and stained for active caspase-3;

FIG. 6J is an immunoblot of lysates from HeLa cells either untreated (C)or treated with 1 μM RA190 (190) or bortezomib (Bz);

FIG. 6K is an immunoblot of lysates from MM1.S cells either untreated(C) or treated with 0.5 μM RA190 or bortezomib for 6 hr;

FIG. 6L is a graph showing expression of surface HSP90 on HeLa cellstreated with 1 mM RA190, bortezomib, or cisplatin as a function of time.

FIG. 7A is a graph showing inhibition of protease function in micefollowing treatment with RA190;

FIG. 7B is a graph showing inhibition of protease function in micefollowing treatment with RA190;

FIG. 7C is a graph showing inhibition of protease function in micefollowing treatment with RA190;

FIG. 7D is a graph showing inhibition of protease function in micefollowing treatment with RA190;

FIG. 7E is a graph showing inhibition of protease function in micefollowing treatment with RA190;

FIG. 8A shows a graph showing inhibition of tumor growth and reductionof tumor size in mice carrying NCI-H929-GFP-luc human tumor cells as afunction of treatment with RA190;

FIG. 8B shows representative bioluminescence images of mice in (FIG. 8A)before (top panel) and after (bottom panel) treatment;

FIG. 8C shows a graph showing decrease in bioluminescence ofEΩ-luciferase tumor cells in mice treated with RA190;

FIG. 8D shows representative bioluminescence images of mice in (FIG. 8C)before (upper panel) and after (lower panel) treatment;

FIG. 8D shows a graph of tumor volume reduction in mice treated withRA190;

FIG. 8F is a graph showing the mean number±SD of IFNγ⁺ CD8⁺ T cells per3×10⁵ splenocytes elicited with or without E7 following treatment withRA190;

FIG. 9A shows an analysis of drug combination index (CI) for RA190 andbortezomib;

FIG. 9B shows an immunoblot of lysates from NCI-H929 cells treated with0.5 μM RA190 (190) or 0.1 μM bortezomib (Bz);

FIG. 9C shows a line chart showing proteasome activity as a function oftreatment with RA190 or bortezomib;

FIG. 9D shows a bar graph showing tryptic activity as a function oftreatment with RA190 or bortezomib;

FIG. 9E shows a bar graph showing PGPH activity as a function oftreatment with RA190 or bortezomib;

FIG. 9F shows a bar graph showing the degradation of Ub-AMC;

FIG. 9G shows a bar graph showing the degradation of Ub-AMC;

FIG. 9H shows the effects on the activity of 19S RP as a function oftreatment with corresponding compounds;

FIG. 9I shows an immunoblot showing the effect on proteasome activity byvarious compounds;

FIG. 9J is a graph showing cell viability;

FIG. 9K is a graph showing cell viability;

FIG. 10A shows cell viability as a function of treatment with indicatedcompounds;

FIG. 10B is an immunoblot showing the proteasome activity of cellstreated with the indicated compound;

FIG. 10C is an immunoblot showing the proteasome activity of cellstreated with the indicated compound;

FIG. 10D is an immunoblot of lysates of control cells;

FIG. 10E is an immunoblot of lysates of cells treated with RA190;

FIG. 11A shows ¹H, ¹⁵N HSQC spectra of ¹⁵N labeled RPN13 and afterincubation with RA190;

FIG. 11B shows ¹H, ¹⁵N HSQC spectra of ¹⁵N labeled RPN13 Pru domain andafter incubation with RA190;

FIG. 11C shows ¹⁵N HSQC spectra of ¹⁵N labeled RPN13 UCH37-bindingdomain and after incubation with RA190;

FIG. 11D shows ¹H, ¹⁵N HSQC spectra of ¹⁵N-labeled RPN13 Pru and afterincubation with RA190 and 5 mM β-mercaptoethanol;

FIG. 11E shows ¹H, ¹⁵N HSQC spectra of ¹⁵N labeled RPN13 Pru domain withall of its native cysteines replaced with alanine (RPN13 Pru C/A) andfollowing incubation with RA190;

FIG. 11F shows ¹H, ¹⁵N HSQC spectra of ¹⁵N labeled RPN13 Pru domain withC88 replaced with alanine (RPN13 Pru C88A) and following incubation withRA190;

FIG. 11G shows a graph of a LC-MS experiment on RA190-exposed RPN13 PruC88A;

FIG. 12A shows a first model of the human RPN13 Pru˜RA190 interaction;

FIG. 12B shows a second model of the human RPN13 Pru˜RA190 interaction;

FIG. 12C shows a third model of the human RPN13 Pru˜RA190 interaction;

FIG. 12D shows a chemical structure of RA190 and RPN13 C88 side chain;

FIG. 12E shows a model of the RPN13 structure highlighting the aminoacids from (12F) that shift after RA190 incubation (T273, L314 and M342)in green and C88 in yellow;

FIG. 12F shows expanded regions of HSQC spectra recorded on RPN13 (toppanels) and after incubation with 10-fold molar excess RA190 (secondpanels) and of RPN13 (253-407, ‘RPN13 CTD’, third panels). A merger ofthe three upper panels is displayed in the bottom panels;

FIG. 13A shows the viability of cells with two, one or no alleles ofTP53 or with mutations to TP53 following treatment with varyingconcentrations of RA190;

FIG. 13B shows a western blot analysis of lysates from HeLa cellstreated with RA190 or bortezomib. Blots were stained with antibodiesagainst p21 and Puma.

FIG. 14 shows a graph showing the pharmacokinetics of RA190 given tomice i.p. or orally;

FIG. 15A shows a western blot showing levels of poly-ubiquitinatedproteins in lysates of tumor cells from mice treated with RA190;

FIG. 15B shows a graph showing the weight of mice as function oftreatment with RA190;

FIG. 16A shows the efficacy of select compounds against HC1806 breastcancer cells;

FIG. 16B shows the efficacy of select compounds against HS578T breastcancer cells;

FIG. 16C shows the efficacy of select compounds against BT549 breastcancer cells;

FIG. 16D shows the efficacy of select compounds against MB-231 breastcancer cells;

FIG. 16E shows streptavidin peroxidase-probed blot in which RA190Acrcompetes the binding of RA190B to RPN13 in Triple Negative Breast Cancer(HS578T) cell lysate;

FIG. 17 shows a representative HPLC trace for RA190 purification;

FIG. 18 shows a graph showing the effect of various compounds on HeLacell viability;

FIG. 19 shows a graph showing a 20S proteasome inhibition assay;

FIG. 20 shows a graph showing the effect of various compounds onNF_(K)B;

FIG. 21A shows an immunoblot of PAO3C cell lysates;

FIG. 21B shows an immunoblot showing the effect of RA190 on thepoly-ubiquitinated protein levels;

FIG. 21C shows an immunoblot showing the effect of RA190 on the levelsof apoptotic proteins and activated caspase-3;

FIG. 21D shows an immunoblot showing the effect of RA190 on CDKinhibitor p27;

FIG. 21E shows a data graph showing the effect of RA190 on Annexin Vpositive cells. 21F shows a data graph showing the effect of RA190 onactive-caspase-3;

FIGS. 22A-D show a series of data graphs showing the effect of indicatedcompounds on the viability of various cancer cell lines;

FIG. 23 shows streptavidin peroxidase-probed blot showing that RA195binds covalently to RPN13. HeLa cell lysate was incubated withcorresponding compounds (RA183 and RA183 B=20 μM; RA195=20 μM;RA195B=10, 20, 50 μM) for 1 hour at 4° C. and subjected to SDS-PAGE,transfer to a PVDF membrane and probed using HRP-Streptavidinperoxidase. RA195B labels HeLa cell lysate at 42 KDa which disappearswith the pretreatment of RA195 indicates that RA195 also binds to RPN13;

FIG. 24 shows an immunoblot showing that RA195 causes accumulation ofPoly-Ubiquitinated proteins (left) and elevation of apoptotic proteinBax (right);

FIG. 25 shows a data graph showing that RA195 and analogs stabilize4UBFL(tetraubiquitin firefly luciferase). HeLa cells transfected with anexpression vector for 4UBFL were then treated with RA195 or its analogsRA195Ac and RA195Bn for 4 hours and Firefly Luciferase activitymeasured;

FIG. 26 shows a series of data graphs showing the effect of RA195 on theexpression of active caspase-3 as determined by flow cytometry using aPE-labeled monoclonal antibody specific for the active form ofcaspase-3;

FIG. 27 shows a series of data graphs showing that treatment of NCIH929cells with RA195, RA195AC, 195BN and RA183 caused an increase incaspase-3 expression as determined by flow cytometry using a PE-labeledmonoclonal antibody specific for the active form of caspase-3;

FIG. 28 shows a series of data graphs showing that treatment of U266cells with RA195, RA195AC, 195BN and RA183 caused an increase in activecaspase-3 expression as determined by flow cytometry using a PE-labeledmonoclonal antibody specific for the active form of caspase-3;

FIG. 29 shows a data graph showing that RA195 and its analog 195Acinhibit TNFα-induced NFκB activation. HEK293 cells transientlytransfected with either NFκB/FL (firefly luciferase reporter constructunder control of an NFκB-driven promotor) or control CMV promotor-drivenFL reporter construct were treated with compounds and TNFα (10 ng/ml)for 7 h. Upon the addition of luciferin, bioluminescence was measured incell lysates using a luminometer. HEK293 cells showed dose dependentdecrease in NF-κB associated promoter activity after RA195 and RA195Actreatment;

FIG. 30 shows an immunoblot showing that poly-ubiquitinated proteins(poly-UB) accumulated and p53 and p21 protein levels are increased incells treated with RA190 and RA195;

FIG. 31 shows a classical solution phase reaction used to generatevarious compounds.

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided to enable any person skilled inthe art to make and use the invention and sets forth the best modescontemplated by the inventor of carrying out his invention. Variousmodifications, however, will remain readily apparent to those skilled inthe art, since the general principles of the present invention have beendefined herein specifically to provide one example of one application ofthe invention.

Activity of Bis-Benzylidine Piperidone Derivatives

We recently described a series of 1,3-diphenylpropen-1-one(chalcone)-based derivatives bearing a variety of amino acidsubstitutions on the amino group of the 4-piperidone, including RA1,which inhibits ubiquitin-mediated protein degradation and preferentiallykills cervical cancer cells.⁶ The α,β ketone represents the minimalmolecular determinant for inhibition, which occurs without affecting CPactivity.³ To improve activity and solubility, and to probe theirpharmacophore, we generated a series of derivatives with varyingsubstituents in the aromatic ring to modulate the acceptor character ofthe enone system including o- and p-halogens, and different amino acidsat the amine functionality of 4-piperidone. Because our previous worksuggested its importance for proteasome inhibitory activity⁶, most RAcompounds incorporated phenylalanine and/or substituted phenylalanine.To overcome the poor solubility and pharmacokinetics of our previousgeneration molecules, we employed an amide in lieu of a urea linkagebetween amino acids and 4-piperidone. We synthesized RA166 and RA201with chlorine and fluorine at the ortho position of the aromatic ringsand phenylalanine attached to the 4-piperidone, and additional compoundsin which histidine or tyrosine (RA213) are substituted forphenylalanine, as well as RA181 with no substituent. Earlier studiessuggested the value of two chlorine atoms on each phenyl moiety and thuswe synthesized RA190 and RA190Ac, which differ in the phenylalanine andamide conjugation compared to the urea conjugation in our earlygeneration molecule RA1.⁶

Using a variety of cancer cell lines, cell viability was determined withan 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide (XTT) assay after 48-hr treatment with titrations of eachcompound (Table 1). Activity against several cell lines known to besensitive to proteasome inhibitors was observed, including those derivedfrom cervical cancer (HeLa, CaSki, and SiHa), MM (ANBL6,MM.1S, NCI-H929,U266, and RPMI-8226), colon cancer (HCT116), and ovarian cancer (ES2 andOVCAR3; Table 1; FIGS. 1A through 1H). FIGS. 1A-H show that RA190 causesa toxic accumulation of polyubiquinated proteins. FIGS. 1 A-D showpercent cell viability of RPMI-8226, ANBL6, and their respective invitro selected bortezomib-resistant cell lines RPMI-8226-V₁₀R andANBL6-V₁₀R as a function of 48 hr treatment with the indicatedcompounds. FIGS. 1 E-H show percent cell viability of indicated MM celllines as a function of 48 hr treatment with the indicated compounds.Because RA190 consistently exhibited the most potent antiproliferativeeffects against MM lines (half maximal inhibitory concentration[IC50]≤0.1 μM) and HPV transformed cells (IC50≤0.3 μM), it was the focusfor further analysis. RA190 was less efficacious against HPV− (IC50>5 μMfor HT3 and C33A, Table 1) than HPV+ (HeLa, CaSki, and SiHa) cervicalcancer cell lines. Likewise, the HPV16-immortalized oral keratinocyteline HOK-16B was more sensitive to RA190 than either HaCaT cells (HPV−,spontaneously immortalized keratinocytes) or FaDu (HPV− head and neckcancer cells).

TABLE 1 Impact of bis-benzylidine piperidone derivatives and proteasomeinhibitors on the viability of cell lines (IC50 values in μM). CompoundCell Lines (RA-) HeLa CasKi SiHa HT3 C33A FaDu HOK-16B SKOV3 OVCAR3 ES-2MM.1S NCl-H929 RPMI8226 HaCaT 166 0.6 1.5 2.0 >5 >5 >5 >5 >2.5 >5 NT NTNT NT >2.5 181 0.35 2.0 2.0 >5 >5 >5 >5 >2.5 >5 NT NT NT NT >2.5 1900.15 0.3 0.75 >5 >5 >2.5 0.6 >2.5 2.0 0.75 0.035 0.05 0.10 >2.5 196 0.61.0 2.0 >5 >5 >5 3.0 >2.5 2.5 NT NT NT NT >2.5 201 0.35 2.5 2.0 >5 >5 >51.5 >2.5 >5 NT NT NT NT >2.5 213 0.6 0.6 2.5 >5 >5 >5 1.5 >2.5 >5 NT NTNT NT >2.5 190Ac 0.17 0.3 1.0 >5 >5 >2.5 0.75 >2.5 2.0 NT 0.05 0.0750.15 >2.5 Bortezomib 0.03 0.05 1.0 <2.5 <2.5 <2.5 0.02 <2.5 <2.5 0.020.005 0.005 0.01 <2.5 MG132 0.75 0.5 2.0 >5 >5 >2.5 0.7 >2.5 NT NT NT NTNT >2.5

MM cells may acquire bortezomib resistance by severalmechanisms.^(22,28) We tested RA190 potency against two MM cell linesthat developed resistance after extended culture in bortezomib,²² and itwas equally efficacious against both the bortezomib-resistant derivativelines and the parental lines, consistent with a mode of action distinctfrom bortezomib (FIGS. 1A-D). Furthermore, the combination of RA190 andbortezomib provides a synergistic effect on the loss of cervical cancercell viability (FIG. 9A). In FIG. 9A, HeLa cells were treated with RA190(0-300 nM) and/or bortezomib (0-30 nM) alone or in combination for 48hr, and cell viability determined. The combination indices and anormalized isobologram were calculated using Compusyn software with IC50of 184 nM for RA190 and 21.7 nM for bortezomib.

RA190 Triggers Accumulation of Polyubiquitinated Proteins

FIGS. 1A-1F show that RA190 causes a toxic accumulation ofpolyubiquitinated proteins. FIGS. 1 A-D show percent cell viability ofRPMI-8226, ANBL6, and their respective in vitro selectedbortezomib-resistant cell lines RPMI-8226-V₁₀R and ANBL6-V₁₀R as afunction of 48 hr treatment with the indicated compounds. FIGS. 1 E-Hshow percent cell viability of indicated MM cell lines as a function of48 hr treatment with the indicated compounds. FIG. 11 is a western blotof lysates from HeLa cells treated with RA190 (190), RA190ME (190ME), orbortezomib (Bz) for 4 hr (left) or 12 hr (right) at the concentrationsindicated. FIG. 1J shows luciferase activity of HeLa cells transientlytransfected with either tetra-ubiquitin-fused firefly luciferase (4UbFL)or FL plasmids as a function of proteasome inhibition by variouscompounds. Because compounds related to RA190 are proteasomeinhibitors,³ we examined its impact on the levels of polyubiquitinatedproteins in HeLa and CaSki cells by anti-K48-linked ubiquitin immunoblotanalysis. RA190 treatment of HeLa cells (4 hr) dramatically increasedthe levels of K48-linked polyubiquitinated proteins similarly tobortezomib (FIG. 11) and in a dose-dependent manner. However,accumulated K48 polyubiquitinated proteins observed following exposureto RA190 exhibited a higher molecular weight than that seen inbortezomib-treated cells (FIG. 11) and occurred more rapidly (FIG. 9B).In FIG. 9B, lysates from NCI-H929 cells treated with 0.5 μM RA190 (190)or 0.1 μM bortezomib (Bz) for the indicated periods of time (left) orfrom CaSki cells treated with DMSO or RA190 for 12 hr (right) weresubjected to immunoblot analysis using anti-Ubiquitin antibody. Actinwas used as a loading control. These results suggest that the toxicityexerted by RA190 for cervical cancer cells is associated with a prioraccumulation of high-molecular-weight polyubiquitinated proteins andoccurs by a mechanism distinct to bortezomib. Indeed, unlike bortezomib,RA190 does not inhibit CP chymotryptic, tryptic, and PGPH activities(FIGS. 9C-E). In FIG. 9C-E, purified 20S proteasomes were treated for 30min with or without compounds (1 μM) prior to the addition of thespecific fluorogenic substrate for chymotryptic (9C), tryptic (9D) orPGPH (9E) hydrolytic proteasome capacities. Mean±SD fluorescenceassociated with AMC released from the substrate was measured at 45 min.Inhibition of RP deubiquitinase activity can produce a similaraccumulation of high-molecular weight polyubiquitinated protein as seenfor RA190.²¹ However, the degradation of Ub-AMC by either purifiedrecombinant UCH37 (with or without the addition of RPN13) or purified RPwas minimally affected by RA190, suggesting that it does not inhibit theRP deubiquitinases (FIGS. 9F-H). In FIGS. 9F and 9G, hydrolysis ofubiquitin—AMC (0.5 μM) alone (grey), or with 20 nM RPN13 (brown), 2 nMUCH37 (black), a mixture of 2 nM UCH37 and 20 nM RA190 (blue), a mixtureof 2 nM UCH37 and 20 nM RPN13 (red), and a mixture of 2 nM UCH37, 20 nMRPN13 and 20 nM RA190 (yellow) monitored by fluorescence (JASCO FP-6200spectrofluorometer; λex=380 nm, λem=460 nm). DMSO was added to allsamples to a final concentration of 0.1% (upper panel). The effect ofDMSO is highlighted by including ubiquitin—AMC (0.5 μM) hydrolysis with2 nM UCH37 lacking DMSO (orange, bottom panel). In FIG. 9H, purified 19SRP (500 nM) in buffer was treated with corresponding compounds 30 min at37° C. and AMC cleavage was measured with a fluorometer for 20 min. Ubalwas used as a positive control.

RA190 Stabilizes Tetraubiquitin-Fused Luciferin

A tetraubiquitin-firefly luciferase (4UbFL) reporter, in which fourcopies of ubiquitin (G76V) are genetically fused to the N terminus offirefly luciferase (FL), is rapidly degraded by the proteasome whereasFL alone has a much longer half-life. Importantly, treatment of cellsexpressing 4UbFL with proteasome inhibitors results in its stabilizationand an increase in luciferase activity, providing a validated approachto assess proteasome function in live cells.²⁴ Two days aftertransfection with either 4UbFL or FL expression vectors, HeLa cells weretreated for 4 hr with bortezomib and luciferase-driven bioluminescencewas dramatically increased in cells expressing 4UbFL but not FL (FIG.1J). Thus, the ratio of bioluminescence observed in cells transfectedwith 4UbFL versus FL was used to assess the proteasome inhibition by theactive compounds in the series, revealing RA190 as more potent thanothers, including RA166 and RA201 (FIG. 1J).

RA190 Covalently Binds to the RP Subunit RPN13

Removal of the olefin bond from RA190 by treatment withβ-mercaptoethanol (forming RA190ME) significantly reduced its potency inboth cell killing and polyubiquitin accumulation assays (FIGS. 1I and9I), suggesting it is a Michael acceptor with olefin bonds that aresusceptible to nucleophilic attack. In FIG. 9I, HeLa cells were treatedwith DMSO, RA190, RA190ME, RA190R, RA1900 and RA190D at 1 μMconcentration for the period of 4 hr and the cell lysate was subjectedto immunoblot analysis with anti-Ubiquitin antibody (left panel). SiHacells were treated with the indicated compounds for 12 hr and the lysatesubjected to Western blot analysis with anti-Ubiquitin antibody (rightpanel). Actin was used as loading control. Elimination of the enonemoiety (RA190R) or conversion of the carboxyl moiety to oxime (RA1900)dramatically reduced activity in cell killing and functional assays(FIG. 9I). Furthermore, washout studies are also consistent with RA190acting as an irreversible inhibitor (FIGS. 9J and 9K). In FIGS. 9J and9K, HeLa cells were either continuously incubated for 24 hr withbortezomib or RA190, (+/+), or after a 1 hr exposure period, incubatedwith fresh inhibitor-free medium for an additional 23 hr (+0, whereuponcell mean viability±SD was determined.

To identify its cellular target, biotin was covalently linked to RA190via its free amine functionality (RA190B). Biotinylation of RA190 didnot affect its potency in cell killing and functional assays (FIGS.10A-10C). FIGS. 10 A-10E show a series of graphs and immunoblots showingthe effects of several compounds on cell viability and proteasomeactivity. 10A shows cell viability as a function of treatment withindicated compounds. 10B and 10C show immunoblots of the proteasomeactivity of cell lines treated with the indicated compounds. 10D and 10Eshow immunoblots of lysates of 293 cells treated for 1 hr at 4° C. withcontrol (DMSO, 10D) or RA190 (10E). In FIG. 10A, HeLa cells were treatedwith RA190, RA190B or RA190R for 48 hr and mean cell viability±SD wasdetermined. IC50 values were determined in triplicate. In FIG. 10B, HeLacells were treated for 12 hr and the cell lysate was subjected toimmunoblot analysis with anti-Ubiquitin antibody. In FIG. 10C, SiHacells were treated with the indicated compounds for 12 hr and the lysatesubjected to Western blot analysis with anti-Ubiquitin antibody. HeLacell lysate was treated with RA190B, subjected to SDS-PAGE, and probedwith streptavidin-peroxidase following protein transfer to apolyvinylidene difluoride (PVDF) membrane. The streptavidin-peroxidasebound to biotinylated cellular proteins, but a striking new band at 42kDa appeared in RA190B-treated samples (FIG. 2A). FIGS. 2A-2F show aseries of SDS PAGE gels that demonstrate that RA190 covalently binds toRPN 13. 2A shows HeLa cell lysates labeled with RA 190B alone or in thepresence of competitor RA190. 2B shows HeLa cell lysates labeled with500 ng purified 19S proteasome, 10 mM RA190B (190B), and 100 mM RA190(190). 2C shows cell lysates of 293TT cells transfected with plasmidexpressing RPN13, RPN10, UCH37 or HHR23B, or luciferase as a control andlabeled with RA190B (20 μM). 2D shows lysates of IPTG induced anduninduced bacteria transduced with expression vector for RPN13 or L2were labeled with RA190B (20 μM). 2E shows Competition for labeling ofRPN13 expressed in bacterial cell lysate with 200 μM RA190 (190), 20 μMRA190B (190B), or both. 2F shows the membrane from (2D) stripped andreprobed with RPN13 antibody. In FIG. 2A, HeLa cells were treated withRA190, RA190B or RA190R for 48 hr and mean cell viability±SD wasdetermined. IC50 values were determined in triplicate. Importantly,RA190 was competitive for this interaction, suggesting specificity.RA190B bound to the 42 kDa protein in a purified RP preparation, and theinteraction was similarly competed by RA190 (FIG. 2B).

Within the RP there are four proteins (intrinsic ubiquitin receptorsRPN10 and RPN13, deubiquitinase UCH37, and shuttling ubiquitin receptorHHR23B) with a molecular weight similar (37-45 kDa) to the cellulartarget of RA190. These proteins were overexpressed separately in 293TTcells, and the lysates labeled with RA190B and probed withstreptavidin-peroxidase. Enhanced labeling of a specific band withRA190B was observed in cell lysates overexpressing RPN13, but not theothers (FIG. 2C).

To eliminate the possibility that another RP component was required forRA190B interaction, RPN13 or an irrelevant protein (L2) wasoverexpressed in bacteria. Cell lysate harvested from bacteria eitherwith or without isopropylthio-Malactoside induction of ectopic proteinexpression was labeled with RA190B and probed by blotting withstreptavidin-peroxidase. RA190B reacted strongly with a 42 kDa proteinonly in lysates of bacteria expressing RPN13 (FIG. 2D). Furthermore,this interaction was competed with by unlabeled RA190 and the presenceof RPN13 was confirmed by western blot with RPN13-specific monoclonalantibody (FIGS. 2E and 2F). These findings suggest that RA190 covalentlybinds directly to RPN13. However, glycerol gradient separation studiesindicate that RA190 does not displace RPN13 from proteasome (FIGS. 10Dand E). In FIGS. 10D and 10E, lysates of 293 cells treated for 1 hr at4° C. with control (DMSO, FIG. 10D) or 20 μM RA190 (FIG. 10E) weresubjected to 10-40% (v/v) glycerol gradient centrifugation for 15 hr at4° C. 800 μL fractions were collected and alternate fractions(1,3,5,7,9,11,13,15,17) were analyzed for the presence of proteasomalproteins (RPN13, RPN2, RPN1 and UCH37) by Western blot analysis.

RA190 Ligates to RPN13 Pru Domain

RPN13 contains an N-terminal Pru (pleckstrin-like receptor forubiquitin) domain that binds to ubiquitin^(19,31) and the RP,^(16,17,20,31) and a C-terminal domain that recruits UCH37 to theproteasome.^(17,27,37) We used nuclear magnetic resonance (NMR) todetermine whether RA190 targets a specific RPN13 functional domain.Unlabeled RA190 was incubated overnight at 10-fold molar excess and 4°C. with ¹⁵N-labeled RPN13; ¹⁵N-labeled RPN13 (1-150), which includes itsPru domain; or 15N-labeled RPN13 (253-407), which includes itsUCH37-binding domain. Unreacted RA190 was removed by dialysis andheteronuclear single quantum coherence (HSQC) spectra were acquired toevaluate the effect of RA190 on the three RPN13 constructs. The spectrumacquired on full-length RPN13 after incubation with RA190 exhibitedsignificant signal loss for specific amino acids in its Pru domain, butnot its UCH37-binding domain (FIGS. 3A and 11A). Moreover, RA190significantly affected NMR spectra recorded on the RPN13 Pru domain(FIGS. 3B and 11B), but not RPN13 (253-407; FIG. 11C). These dataindicate that RA190 interacts with the RPN13 Pru domain. FIG. 3 FIGS.3A-3G show a series of figures that demonstrate how RA190 interacts withthe RPN13 Pru Domain. 3A and 3B show enlarged regions of HSQC spectrafor ¹⁵N-labeled human RPN13 (3A, black) or RPN13 Pru domain (3B, black)and after RA190 incubation (3A and 3B, orange). 3C-3F shows graphs ofLC-MS experiments for RPN13 Pru domain (3C) and RA190-exposed RPN13(3D), RPN13 Pru domain (3E), and RPN13 Pru C^(60,80,121) A (3F). 3Gshows HSQC spectra of ¹⁵N-labeled RPN13 Pru C^(60,80,121) A (black) andafter RA190 incubation (orange). FIGS. 11A-11G show a series of spectradata showing that RA190 binds specifically to RPN13 Pru domain andrequires its C88 and no reducing agent. 11A shows ¹H, ¹⁵N HSQC spectraof ¹⁵N labeled RPN13 (black) and after incubation with RA190 (orange).11B shows ¹H, ¹⁵N HSQC spectra of ¹⁵N labeled RPN13 Pru domain (black),and after incubation with RA190 (orange). 11C shows ¹⁵N HSQC spectra of¹⁵N labeled RPN13 UCH37-binding domain (black), and after incubationwith RA190 (orange). 11D shows ¹H, ¹⁵N HSQC spectra of ¹⁵N-labeled RPN13Pru (black) and after incubation with RA190 and 5 mM β-mercaptoethanol(orange). 11E shows ¹H, ¹⁵N HSQC spectra of ¹⁵N labeled RPN13 Pru domainwith all of its native cysteines replaced with alanine (RPN13 Pru C/A,black) and following incubation with RA190 (orange). 11F shows ¹H, ¹⁵NHSQC spectra of ¹⁵N labeled RPN13 Pru domain with C88 replaced withalanine (RPN13 Pru C88A, black) and following incubation with RA190(orange). 11G shows a graph of a LC-MS experiment on RA190-exposed RPN13Pru C88A.

β-mercaptoethanol prevented the effect of RA190 on the ¹⁵N-labeled RPN13Pru domain (FIG. 11D). FIG. 11D shows ¹H, ¹⁵N HSQC spectra of¹⁵N-labeled RPN13 Pru (black) and after incubation with RA190 and 5 mMβ-mercaptoethanol (orange). To test whether RA190 interacts covalentlywith the RPN13 Pru domain, we compared liquid chromatographyhigh-resolution mass spectra acquired on our ¹⁵N-labeled RPN13 sampleswith and without RA190 incubation (FIGS. 3C-3F). Unmodified RPN13 waspresent in each of the samples exposed to RA190 along with an additionalspecies at a molecular weight shifted by 561.4 Da for the full-lengthprotein (FIG. 3D) and 559.8 Da for the RPN13 Pru domain (FIG. 3E); theexpected molecular weight shift caused by RA190 attachment is 561.31 Da.Thus, one RA190 molecule adducted to the RPN13 Pru domain.

RA190 Adducts to RPN13 C88

We used site-directed mutagenesis and NMR to determine the site of RA190ligation. RA190 no longer interacts with RPN13 Pru domain when its fournative cysteines are replaced with alanine (RPN13 Pru C/A). ¹⁵N-labeledRPN13 Pru C/A incubated with RA190 exhibited no changes in HSQCexperiments compared to RPN13 Pru C/A alone (FIG. 11E). FIG. 11E shows¹H, ¹⁵N HSQC spectra of ¹⁵N labeled RPN13 Pru domain with all of itsnative cysteines replaced with alanine (RPN13 Pru C/A, black) andfollowing incubation with RA190 (orange).

Inspection of RPN13 NMR spectra acquired with and without RA190 revealedthat C88 was significantly affected (FIGS. 3A and 3B), and we testedwhether this cysteine is required for the interaction. RA190 did notcause changes to NMR spectra recorded on 15N-labeled RPN13 Pru C88A(FIG. 11F), and only one species of the correct molecular weight forRPN13 Pru C88A was observed by mass spectrometry (FIG. 11G). Bycontrast, RPN13 Pru C^(60,80,121). A, in which only C88 was preserved,exhibited significant spectral changes upon incubation with RA190 (FIG.3G) and a molecular weight shift of 559.9 Da by MS (FIG. 3F). These dataindicate that RA190 ligates to RPN13 C88.

Model of RPN13 Pru Adducted with RA190

We quantified the RA190 effect on RPN13 Pru by integrating the NMRsignal of spectra acquired on free and RA190-adducted RPN13 Pru (FIGS.3B and 11B). The ratio of these values was plotted for each backbone(FIG. 4A) and side chain (FIG. 4B) amide group. FIGS. 4A-4C show a pairof bar graphs and a model showing the amino acid residues implicated inthe RA190 and RPN13 interaction. 4A and 4B show graphs of normalizedpeak intensity attenuation of RPN13 Pru domain backbone (4A) and sidechain (4B) amide groups upon binding RA190. 4C shows the lowest energymodeled structure for human RPN13 Pru˜RA190. In FIGS. 4A and 4B, thedashed line indicates one SD above average. Unassigned, overlapping, orproline groups are excluded from this analysis and indicated (*). Thisanalysis highlighted RPN13 Pru amino groups that are significantlyaffected by RA190 and was used to generate model structures of RPN13 Pruadducted with RA190 in HADDOCK.¹⁵ The amino acids most affected by RA190map to a region opposite RPN13 ubiquitin binding loops that includesC88. Despite its small size and covalent attachment, RA190 addition toRPN13 led to signal loss (FIGS. 3A and 3B), which suggests that it mayadopt multiple configurations when adducted to RPN13. Our structurecalculations yielded four major RA190 conformations when adducted toRPN13 C88 Sg (FIGS. 4C and 12A-12C). FIGS. 12A-12F show a series ofmodels and graphs showing detailing the interaction between RA190 andRPN13. 12A-12C show models of the human RPN13 Pru˜RA190 interaction. 12Dshows a chemical structure of RA190 and RPN13 C88 side chain. 12E showsa model of the RPN13 structure highlighting the amino acids from (12F)that shift after RA190 incubation (T273, L314 and M342) in green and C88in yellow. 12F shows expanded regions of HSQC spectra recorded on RPN13(top panels) and after incubation with 10-fold molar excess RA190(second panels) and of RPN13 (253-407, ‘RPN13 CTD’, third panels). Amerger of the three upper panels is displayed in the bottom panels withRPN13, RPN13 with RA190, and RPN13 (253-407) in black, orange, and bluerespectively. In FIG. 4C and FIGS. 12A-12C, amino acids most affected byRA190 are highlighted in darkest red. RA190 carbon, nitrogen, oxygen,and chlorine atoms are colored light blue, indigo, red, and green,respectively. For depiction, we selected the lowest energy structure(FIG. 4C), which was also most consistent with our NMR data (FIGS. 4Aand 4B). Rpn13 UCH37-binding domain abuts the Pru domain,¹¹ and theregion targeted by RA190 is within the interdomain contact surface (FIG.12D). In FIG. 12F, four distance restraints defined between RPN13 C88 Syand RA190 CAI, HAI, CAZ, and CBC atoms are highlighted with arrows andwere used in the structure calculations. An NMR spectrum from a mixtureof RA190-modified and unmodified RPN13 provides evidence that the RPN13interdomain interactions are abrogated by RA190 (FIG. 12F). FIG. 12Fshows expanded regions of HSQC spectra recorded on RPN13 (top panels)and after incubation with 10-fold molar excess RA190 (second panels) andof RPN13 (253-407, ‘RPN13 CTD’, third panels). A merger of the threeupper panels is displayed in the bottom panels with RPN13, RPN13 withRA190, and RPN13 (253-407) in black, orange, and blue respectively.

RA190 Causes Endoplasmic Reticulum Stress

The accumulation of unfolded proteins in the endoplasmic reticulumtriggers the UPR, which attempts to restore homeostasis by translationattenuation and upregulation of chaperones. However when the UPR failsto restore homeostasis, it promotes apoptosis. Proteasome inhibitioncreates endoplasmic reticulum stress by blocking the removal ofmisfolded proteins, enhancing IRE1a-mediated splicing of the mRNA codingfor active transcription factor XBP1, one of the main UPR branches, andelevating expression of activating transcription factor-4 (ATF-4) andC/EBP-homologous protein (CHOP)-10, both transcription factors drivingapoptosis. Treatment of MM.1S and HeLa cells with RA190 causedupregulation of ATF-4 protein levels and CHOP-10 and XBP1s mRNA levelsprior to apoptosis (FIGS. 5A-5E). FIGS. 5A-5H show a series ofimmunoblot assays showing that RA190 u[regulates UPR and targets of HPVE6. 5A shows an immunoblot analysis for ATF-4 and actin in MM.1S cellseither untreated (C), or treated with RA190 (190) or bortezomib (Bz) forthe indicated times. 5B shows an immunoblot analysis for ATF-4 in HeLacells either untreated (C), or treated with 1 μM of RA190 (190) or 1 μMbortezomib (Bz) for 6 hr. 5C and 5D show mRNA levels of CHOP-10expression in ATF-4 HeLa cells as a function of a 3 hr (5C) or 12 hr(5D) treatment with RA190 or bortezomib. 5E shows mRNA levels of XBP1expression in ATF-4 HeLa cells as a function of treatment with RA190 orbortezomib. 5F shows an immunoblot analysis for Bax protein levels inMM.1S cells treated as in 5A. 5G shows immunoblot analysis for p53 andβ-tubulin in the indicated cell line either untreated (C) or treatedwith 1 μM RA190 (190) or Bortezomib (Bz) for 24 hr (top panel) or forthe indicated times (bottom panel). 5H shows immunoblot assays for p21,Puma, Bax, Bak, and hDLG-1 at the time points indicated. Specifically,FIG. 5A shows an immunoblot analysis for ATF-4 and actin in MM.1S cellseither untreated (C), or treated with RA190 (190) or bortezomib (Bz) forthe indicated times. FIG. 5B shows an immunoblot analysis for ATF-4 inHeLa cells either untreated (C), or treated with 1 μM of RA190 (190) or1 μM bortezomib (Bz) for 6 hr. FIGS. 5C and 5D show mRNA levels ofCHOP-10 expression in ATF-4 HeLa cells as a function of a 3 hr (5C) or12 hr (5D) treatment with RA190 or bortezomib. FIG. 5E shows mRNA levelsof XBP1 expression in ATF-4 HeLa cells as a function of treatment withRA190 or bortezomib. Bax is a critical element in the induction ofapoptosis by UPR, and RA190 treatment of MM.1S cells significantlyelevated Bax protein levels (FIG. 5F). Conversely, UPR induced celldeath is typically p53-independent. Isogenic HCT116 cells in which bothalleles of wild-type TP53 had been eliminated by homologousrecombination, or HCT-116 cells into which mutant p53 was introduced,exhibited similar sensitivity to bortezomib and RA190 as the parentalline (FIG. 13A), consistent with p53-independent cell death in responseto an unresolved UPR. In FIG. 13A, HCT116 cells containing two WT TP53alleles (+/+), one WT TP53 allele (+/−), or neither (−/−), or a mutantTP53 248R allele with one (248R1+) or no WT TP53 allele (248R1-) werecultured for 48 hr in the presence of the concentrations of RA190indicated and their mean viability±SD was determined. FIGS. 13A-13B showa graph and immunoblot showing cell viability as a function of thepresence, absence or mutations of TP53 and the effect of indicatedcompounds on cellular activities, respectively. 13A shows the viabilityof cells with two, one or no alleles of TP53 or with mutations to TP53following treatment with varying concentrations of RA190. 13B shows awestern blot analysis of lysates from HeLa cells treated with RA190 orbortezomib. Blots were stained with antibodies against p21 and Puma.

RA190 Elevates p53 and p53-Regulated Genes in Cervical Cancer Cells

HPV E6-mediated degradation of p53 and other targets via the proteasomeis a hallmark of high-risk HPV types and critical totransformation,^(10,31) suggesting that stabilization of E6 targets andconsequent pro-apoptotic signaling may account for the greatersensitivity of HPV-transformed cells to RA190. However, combination ofRA190 and bortezomib was synergistic (CI=0.4) for killing of HeLa cellsin vitro, suggesting distinct targets (FIG. 9A). We thereforeinvestigated whether RA190, like bortezomib, could restore the levels ofwild-type p53 in HPV-transformed cervical cancer cells.²³ Treatment ofHeLa, CaSki, and SiHa cells for 24 hr with RA190 elevated p53 levels aswith bortezomib (FIG. 5G, top panel). Rapid and time-dependent recoveryof p53 levels was observed within 2 hr of RA190 treatment, reachingmaximal levels by 6 hr (FIG. 5G, bottom panel). p53-targets p21 andPuma,³⁵ including their ubiquitinated forms, were also increased in atime-dependent manner (FIGS. 5H and 13B). In FIG. 13B, HeLa cells weretreated with RA190 (1 μM) or Bortezomib (1 μM) for the indicated timeperiods and the cell lysate was subjected to Western blot analysis andprobed with anti-p21 and anti-Puma antibody. Tubulin was used as aloading control. Treatment of HeLa cells with RA190 or bortezomibincreased levels of pro-apoptotic factors targeted by E6 fordegradation,²⁶ notably and Bak (FIG. 5H), and the tumor suppressorhDLG-1 in HeLa (FIG. 5H) and CaSki cells (not shown). Thus RA190stabilizes multiple E6 targets, ²⁶ including pro-apoptotic and tumorsuppressor proteins, through proteasome inhibition.

RA190 Induces Apoptosis and Display of HSP90 on the Surface of DyingTumor Cells

The rapid upregulation of pro-apoptotic factors and loss of viabilityupon RA190 treatment may reflect apoptosis. Annexin-V flow cytometricmeasurements made 12 hr after treating MM and HeLa cells indicate thatRA190 and bortezomib trigger extensive apoptosis (FIGS. 6A-6D and6E-6G). FIGS. 6A-G show a series of immunoblots and graphs showing thatRA190 triggers apoptosis and cell surface presentation of HSP90. 6A-6Dshow graphs showing Annexin-V expression in HeLa cells untreated (6A) ortreated with RA190 (6B), RA190ME (6C) or bortezomib (6D). 6E-6G showgraphs showing Annexin-V expression in MM.1S cells untreated (6E) ortreated with RA190 (6F) or bortezomib (6G). 6H shows the amount ofactive caspase-3 in HeLa as a function of treatment with varyingconcentrations of RA190 or bortezomib. 6I is a representative flowcytometry analysis of MM.1S cells treated as in (6E-6G) and stained foractive caspase-3. 6J is an immunoblot of lysates from HeLa cells eitheruntreated (C) or treated with 1 μM RA190 (190) or bortezomib (Bz). 6K isan immunoblot of lysates from MM1.S cells either untreated (C) ortreated with 0.5 μM RA190 or bortezomib for 6 hr. 6L is a graph showingexpression of surface HSP90 on HeLa cells treated with 1 mM RA190,bortezomib, or cisplatin as a function of time. Activation of ICE familymembers such as caspase-3 and -7 results in cleavage of poly ADP ribosepolymerase (PARP) to 85 kDa and 25 kDa fragments and drives apoptosis.The ability of RA190 to trigger caspase-3 activity (FIGS. 6H and 6I) andPARP cleavage (FIGS. 6J and 6K) in MM lines and cervical cancer isconsistent with induction of apoptotic cell death.

Bortezomib treatment of MM cells elevates expression and surfaceexposure of HSP90 in association with “immunogenic” cell death.³³ HeLaand CasKi cells were treated with RA190 or bortezomib and cell surfaceHSP90 detected by flow cytometry. RA190 treatment of HeLa cells for 24hr produced cell-surface HSP90 on 54.2% of cells, whereas 12.8% ofbortezomib-treated cells and only 3% of control cells displayed HSP90,demonstrating that this phenomenon is not restricted to MM cells.Notably, cisplatin did not induce surface display of HSP90, suggestingthat not all types of killing affect cells in this way (FIG. 6L). A timecourse experiment in HeLa cells demonstrated initiation of surface HSP90by 6 hr and strong upregulation by 12 hr following RA190 treatment (FIG.6L), indicating a similar time course to Annexin V-staining (not shown),and more rapid onset than that for bortezomib treatment.

Pharmacokinetics and Safety of RA190

Upon formulation in 20% (w/v) β-hydroxyisopropyl-cyclodextrin in water,mice were treated with various single oral (p.o.) and intraperitoneal(i.p.) doses of RA190. With i.p. administration of 10 mg/kg RA190, peakplasma levels (Cmax) were observed in 2 hr, and then declinedmulti-exponentially with distribution (T_(1/2, α)) and terminal(T_(1/2, β)) half-lives of 4.2 and 25.5 hr, respectively (FIG. 14).After p.o. administration of 20 mg/kg, RA190 plasma concentrations roserapidly during the first hour and then declined exponentially with aT112, R of 2.6 hr. Based on the RA190 AUC values, the bioavailability ofRA190 delivered p.o. relative to i.p. was 7.2%. RA190 was detected inkidney (0.61 μg/g), liver (0.57 μg/g), lung (0.66 μg/g), and spleen(0.59 μg/g), but not brain 48 hr after the mice were given a single i.p.dose of 10 mg/kg RA190. More specifically, FIG. 14 shows mean RA190plasma concentration versus time curves from non-tumor bearing mice(BALB/c mice, 4-6 weeks old) treated with 10 mg/kg intraperitoneally (•)or 20 mg/kg orally (∘). The non-compartmental pharmacokinetic analysisidentified the following parameters; for the 10 mg/kg i.p dose, a Cmaxof 808 ng/mL, an AUC of 4393 ng*hr/mL, T_(1/2),α^(b) of 4.2 hr, aT_(1/2)β^(b) of 25.5 hr, a Ud/F of 83.61 L/kg, a C1/F of 2.3 L/hr/kg;for the 20 mg/kg oral dose, a Cmax of 133 ng/mL, an AUC of 634 ng*hr/mL,an F_(a) of 7.2%, a T_(1/2)βb of 2.6 hr, a Ud/F of 117.3 L/kg, and aC1/F of 31.6 L/hr/kg.

To examine its safety/toxicity profile, mice were given three doses of40 mg/kg RA190 p.o. or vehicle alone every third day and euthanized onday 12. Blood was harvested and blood chemistry and hematologic analysesperformed (Table 2). No significant difference between the panels oftests was observed between the vehicle and RA190-treated groups, exceptfor a small reduction in triglycerides. The histopathology of the lungs,kidney, spleen, and liver was unremarkable in both the vehicle andRA190-treated animals. Similar studies performed in mice bearing TC-1tumors and treated with either RA190 or bortezomib also suggest thatRA190 has a promising safety profile (Table 3).

TABLE 2 Complete blood counts and blood chemistry of healthy Balb/C micetreated with 3 doses of RA190 (40 mg/kg p.o. every third day) for aperiod of 9 days. Vehicle RA190 Complete (mean ± SD, (mean ± SD, NormalBlood Counts n = 3) n = 3) Range Leukocytes WBC(White Blood  9.03 ± 3.857.88 ± 3.42  1.8-10.7 Cells) NE(Neutrophils)  2.37 ± 1.47 2.43 ± 0.940.1-2.4 LY(Lymphocytes)  5.92 ± 1.91 4.78 ± 1.89 0.9-9.3 MO(Monocytes) 0.42 ± 0.20 0.34 ± 0.28 0.0-0.4 EO (Eosinophils)  0.24 ± 0.22 0.24 ±0.23 0.0-0.2 BA(Basophils)  0.07 ± 0.04 0.06 ± 0.07 0.0-0.2 ErythrocytesRBC (red Blood 10.53 ± 0.46 10.38 ± 0.26  6.36-9.42 Cells) Hb(Hemoglobin) 15.26 ± 0.35 14.63 ± 0.15  11.0-15.1 HCT (Hematocrit) 58.30± 1.83 55.66 ± 1.70  35.1-45.4 MCV (Mean 55.36 ± 0.72 53.60 ± 0.26 45.4-60.3 corpuscular Volume) MCH (Mean 14.50 ± 0.4  14.10 ± 0.2 14.1-19.3 corpuscular hemoglobin) MCHC (mean 26.20 ± 0.43 26.33 ± 0.55 30.2-34.2 corpuscular hemoglobin concentration) RDW (Red Blood 17.40 ±0.55 17.23 ± 0.30  12.4-27.0 Cell Distribution Width) Thrombocytes PLT(Platelet) 605.33 ± 81.64  533 ± 61.65  592-2972 MPV (Mean  5.13 ± 0.255.06 ± 0.37  5.0-20.0 Platelet Volume) Blood Chemistry Panel CHOL(Cholesterol) 121.33 ± 13.01  114 ± 12.12  60-165 TRIG 156.33 ± 14.74107.33 ± 22.40  109-172 (Triglycerides) UA (Uric Acid) 1.466 ± 0.11  1.5± 0.26 CK_NEW 89.66 ± 36.6 55.66 ± 25.48 (Creatinine Kinase) GGTNEW 3.66 ± 0.57 4.33 ± 0.57 (Gamma-Glutamyl Transferase) ALTNEW (Alanine35.33 ± 2.08 39.66 ± 4.04  20-80 Aminotransferase) ASTNEW  51 ± 3.062.66 ± 11.01  50-300 (Aspartate aminotransferase) AMYL (Amylase) 890.66± 59.1   743 ± 29.4 1063-1400 ALPNEW (Alkaline  82.66 ± 10.69 84.33 ±7.57  28-96 Phosphatase) TBIL1 (Total  0.26 ± 0.05 0.26 ± 0.05 0.1-0.9bilirubin) GLU (Glucose) 175.66 ± 4.04  175.33 ± 20.5   62-175 TPROT(Total  5.2 ± 0.26 5.16 ± 0.05 3.5-7.2 protein) CA (Calcium)  8.96 ±0.23  9 ± 0.2  9.0-13.0 BUNNEW (Blood 18.66 ± 1.52  20 ± 2.64 17-31 UreaNitrogen) CREAT  0.33 ± 0.05 0.36 ± 0.05 0.3-1.0 (Creatinine) ALBNEW(Albumin)  3.06 ± 0.15 3.03 ± 0.05 2.5-4.8 HDLNEW (High  51 ± 4.0  49 ±5.29 45-96 Density Lipoprotein) LDH (lactate 164.66 ± 18.5   160 ± 37.51Dehydrogenase) Na (Sodium) 148.5 ± 2.12 147.5 ± 0.70  K (Potassium)  6.1± 0.14 6.05 ± 0.21

TABLE 3 Complete blood counts and blood chemistry of C57BL/6 black micecarrying TC-1 tumor treated with nine doses of RA190 (40 mg/kg p.o.every third day) and Bortezomib (1.5 mg/kg i.p. every third day).Vehicle RA190 Bortezomib CBC (Complete (mean ± SD, (mean ± SD, (mean ±SD, Normal Blood Counts) n = 3) n = 3) n = 3) Range Leukocytes WBC(WhiteBlood 13.56 ± 4.21  7.62 ± 4.17 24.85 ± 23.94  1.8-10.7 Cells)NE(Neutrophils) 9.72 ± 5.42 2.69 ± 0.45 24.06 ± 15.07 0.1-2.4LY(Lymphocytes) 3.14 ± 1.34 1.87 ± 0.69 2.12 ± 1.83 0.9-9.3MO(Monocytes) 0.38 ± 0.09 0.25 ± 0.07 0.20 ± 0.13 0.0-0.4 EO(Eosinophils) 0.27 ± 0.15 0.10 ± 0.01 0.64 ± 0.48 0.0-0.2 BA(Basophils)0.04 ± 0.00 0.023 ± 0.011 0.07 ± 0.06 0.0-0.2 Erythrocytes RBC (redBlood 8.84 ± 0.24 9.77 ± 0.76 7.63 ± 1.01 6.36-9.42 Cells) Hb(Hemoglobin) 12.56 ± 0.68  13.26 ± 1.00  11.3 ± 0.70 11.0-15.1HCT(Hematocrit) 45.46 ± 0.55  50.36 ± 2.45  42.36 ± 4.15  35.1-45.4 MCV(Mean 51.40 ± 0.81  51.66 ± 2.63  55.66 ± 1.72  45.4-60.3 corpuscularVolume) MCH (Mean 14.23 ± 0.90  13.60 ± 0.53  14.90 ± 1.05  14.1-19.3corpuscular hemoglobin) MCHC (mean 27.63 ± 1.65  26.33 ± 0.75  26.73 ±1.16  30.2-34.2 corpuscular hemoglobin concentration) RDW (Red Blood18.63 ± 1.12  18.00 ± 0.52  20.13 ± 0.61  12.4-27.0 Cell DistributionWidth) Thrombocytes PLT (Platelet) 886.33 ± 128.0   907 ± 45.92 1346 ±87.5   592-2972 MPV (Mean Platelet 5.50 ± 0.2  5.36 ± 0.06 5.40 ± 0.10 5.0-20.0 Volume) Blood Chemistry Panel CHOL (Cholesterol)   77 ± 11.31 67 ± 4.58  69 ± 7.93  60-165 TRIG (Triglycerides)  54 ± 4.24 61.33 ±1.15  56.66 ± 15.88 109-172 UA (Uric Acid) 2.25 ± 0.21 2.86 ± 0.41 2.73± 0.87 CK_NEW (Creatinine  110 ± 24.04  131 ± 65.50 93.66 ± 3.52 Kinase) GGTNEW (Gamma-  5.5 ± 0.70 5.33 ± 0.57 5.00 ± 0.67 GlutamylTransferase) ALTNEW (Alanine 27.5 ± 0.7  31.33 ± 2.30  26.33 ± 2.51 20-80 Aminotransferase) ASTNEW (Aspartate  47 ± 5.66 50.33 ± 4.93    53± 18.52  50-300 aminotransferase) AMYL (Amylase)  417 ± 74.95 621.33 ±127    473 ± 57.86 1063-1400 ALPNEW (Alkaline 34.50 ± 10.60 55.33 ±6.50   49 ± 5.29 28-96 Phosphatase) TBIL1 (Total bilirubin) 0.2 ± 0.00.233 ± 0.057 0.26 ± 0.05 0.1-0.9 GLU (Glucose) 154.50 ± 28.99  170.66 ±6.11   128 ± 20.29  62-175 TPROT (Total 4.25 ± 0.07 4.46 ± 0.11 4.40 ±0.26 3.5-7.2 protein) CA (Calcium) 8.60 ± 0.28 8.56 ± 0.23 8.83 ± 0.60 9.0-13.0 BUNNEW (Blood 19.0 ± 4.24  26 ± 5.29 22.66 ± 5.13  17-31 UreaNitrogen) CREAT (Creatinine) 0.25 ± 0.07  0.46 ± 0.057 0.33 ± 0.050.3-1.0 ALBNEW (Albumin) 2.35 ± 0.07 2.76 ± 0.23 2.50 ± 0.10 2.5-4.8HDLNEW (High 31.5 ± 3.53 40.33 ± 4.72  28.33 ± 4.61  45-96 DensityLipoprotein) LDH (lactate  215 ± 63.63 172.06 ± 14.29  245.33 ± 96   Dehydrogenase)

Proteasome Inhibition In Vivo by RA190

Naked 4UbFL reporter plasmid DNA was delivered either by gene gun intothe skin or in vivo electroporation in muscle (FIG. 7), and the level ofluciferase in the mouse was assessed before and after drug treatment bybioluminescence imaging. FIGS. 7A-7E show a series of graphs showinginhibition of protease function in mice following treatment with RA190as seen by an increase in the amount of 4UbFL. In 7A, mice received4UbFL DNA via electroporation and were orally administered RA190 andbortezomib. In 7B, mice received 4UbFL DNA intradermally and wereadministered RA190 and bortezomib by i.p. In 7C, mice received 4UbFL DNAvia electroporation and were orally administered RA190. In 7D, micereceived 4UbFL intradermally and were orally administered RA190. In 7E,mice received 4UbFL DNA intradermally and were RA190 was topicallyadministered. To examine whether RA190 was able to inhibit proteasomefunction in vivo, mice (ten per group) were injected i.p. with RA190 (40mg/kg), bortezomib (1.5 mg/kg), or vehicle 1 day after intramuscular(i.m.) electroporation with 4UbFL reporter plasmid (FIG. 7A). At 4 hrafter treatment, mice treated with RA190 exhibited 4-fold higher levelsof bioluminescence compared to vehicle alone, whereas bortezomibelevated it only 2-fold. One day post-treatment, both RA190 andbortezomib-treated animals exhibited a similar 4-fold increase inbioluminescence, suggesting continued inhibition of proteasome function(FIG. 7A). The level of bioluminescence observed in the vehicle-treatedmice decreased steadily over 48 hr, suggesting a slow loss oftransfected cells. At 48 hr after RA190 treatment, the mice still showeda significant increase in bioluminescence despite the short half-life ofRA190 in blood (FIG. 7A), possibly reflecting slow regeneration of newproteasomes or the continued presence of RA190 or active metabolites intissues. Because papillomavirus infections are typically restricted toskin and mucosa, the 4UbFL reporter plasmid was delivered into the skinby gene gun.^(8,34) Similar stabilization of the 4UbFL reporter wasobserved, albeit of shorter duration, suggesting that both RA190 andbortezomib were also active in skin (FIG. 7B).

Oral administration of RA190 increased the bioluminescence produced bymice transduced with 4UbFL plasmid either i.m. by electroporation (FIG.7C) or in skin via gene gun (FIG. 7D). Again, the elevation ofbioluminescence in skin was of shorter duration than that for the i.m.studies, possibly reflecting a more rapid turnover of skin cells, ascompared to transduced muscle cells of electroporated animals. Topicaladministration of RA190 at 4% in Cremophor-EL also stabilized the 4UbFLreporter in skin (FIG. 7E). In FIGS. 7A-7E, the transduced mice weretreated with vehicle, RA190, or bortezomib (ten mice per group)respectively. After the indicated time points of treatment,bioluminescence was measured by injection of luciferin and imaging withan IVIS 200 (fold change±SD, *p<0.05, **p<0.01). RA190 was given i.p.(7A and 7B) or orally (7C and 7D) at 40 mg/kg; bortezomib was given i.p.at 1.5 mg/kg dose (7A and 7B). In FIG. 7E, RA190 was given 4% inCremophor-EL topically.

RA190 Treatment Inhibits Tumor Growth in Mice

NOG (NOD/Shi-scid/IL-2Rγnull) mice carrying an NCI-H929 MM line thatexpresses luciferase received RA190 or vehicle 20 mg/kg/day i.p. dailyfor 7 days. Prior to and at the end of the treatment, mice were imagedfor bioluminescence. RA190 showed potent antitumor activity, even with a3-day break in the middle of treatment (FIGS. 8A and 8B). FIGS. 8A-Dshow the effects of RA190 on tumor growth in mice. 8A shows a graphshowing inhibition of tumor growth and reduction of tumor size in micecarrying NCI-H929-GFP-luc human tumor cells as a function of treatmentwith RA190. 8B shows representative bioluminescence images of mice in(8A) before (top panel) and after (bottom panel) treatment. 8C shows agraph showing decrease in bioluminescence of ES2-luciferase tumor cellsin mice treated with RA190. 8D shows representative bioluminescenceimages of mice in (8C) before (upper panel) and after (lower panel)treatment. 8D shows a graph of tumor volume reduction in mice treatedwith RA190. 8F is a graph showing the mean number±SD of IFNγ⁺ CD8⁺ Tcells per 3×10⁵ splenocytes elicited with or without E7 followingtreatment with RA190. The mice were imaged before and at the end of thetreatment for bioluminescence levels. We have previously described thesusceptibility of human ovarian cancer lines to proteasome inhibition.Nude mice carrying ES2-luciferase tumor i.p. were treated with RA190 orvehicle for 14 days and bioluminescence was imaged weekly. Treatmentwith RA190 significantly inhibited the ES2 tumor growth (FIGS. 8C and8D). Specifically, FIG. 8C shows percentage change of bioluminescence±SDin nude mice bearing ES2-luciferase tumor cells (tumor cells injectedi.p.) receiving 10 mg/kg RA190 (i.p.) or vehicle alone (n=8) every day.Prior to and 7 and 14 days after initiation of treatment, the mice wereimaged for luciferase activity.

C57BL6 mice carrying HPV16 E6 and E7-transformed and syngeneic tumorTC-1, which spontaneously induces E7-specific CD8+ T cell responses,were treated with either RA190 (i.p., 20 mg/kg) or vehicle daily for 7days and the tumors were harvested. Tumor lysates probed by western blotfor polyubiquitinated proteins were dramatically elevated in the RA190treatment group, suggesting RA190 can access solid tumor to blockproteasome function (FIG. 15A). Oral administration to tumor-bearingmice of RA190 (40 mg/kg every third day) significantly inhibited(p<0.001) TC-1 tumor growth as compared to treatment with vehicle alone(FIG. 8E). The final tumor weights after the 14-day treatment periodwere 1,873±180 mg in vehicle treated mice and 727±101 mg in theRA190-treated mice (p<0.001). Weight gain and the spontaneousE7-specific CD8+ T cell response did not differ significantly betweenthe vehicle and RA190-treated mice (FIG. 8F; FIG. 15B). FIGS. 15A-15Bshow an immunoblot and data graph showing the effect of RA190 on levelsof polyubiquitination in tumor cells and the effect of RA190 on theoverall weight of mice, respectively. 15A shows a western blot showinglevels of poly-ubiquitinated proteins in lysates of tumor cells frommice treated with RA190. 15B shows a graph showing the weight of mice asfunction of treatment with RA190.

Compounds Efficacy Against Triple Negative Breast Cancer

To test the efficacy of various RA compound derivatives on the viabilityof breast cancer cells, breast cancer cell lines were treated in vitrowith varying concentrations of selected compounds. As seen in FIGS.16A-16D, the viability of HCC 1806, HS578T, BT549 and MB-231 breastcancer cells, respectively, was severely compromised following treatmentwith various concentrations of indicated compounds for 48 hours,suggesting that Ttriple negative breast cancer cell lines are highlysensitive to proteasome inhibition by these compounds. In addition, FIG.16E also demonstrates that RA190 analog RA190Acr competes the binding ofRA190B to RPN13 in HS578T cell lysate, as detected after SDS-PAGE,blotting to a PVDF membrane and probing with streptavidin-peroxidase.FIGS. 16A-16E are a series of data graphs and a western blot showing theefficacy of select compounds against various breast cancer cell lines.16A shows the efficacy of select compounds against HC1806 breast cancercells. 16B shows the efficacy of select compounds against HS578T breastcancer cells. 16C shows the efficacy of select compounds against BT549breast cancer cells. 16D shows the efficacy of select compounds againstMB-231 breast cancer cells. 16E shows streptavidin peroxidase-probedblot in which RA190Acr competes the binding of RA190B to RPN13 in TripleNegative Breast Cancer (HS578T) cell lysate. RA190Acr is another analogof RA190 and showed potency similar to RA190. Briefly HS578T cell lysatewas incubated with corresponding compounds for the period of 1 hr at 4 Cand subjected to Western blot analysis and probed for HRP-Streptavidin.A new band at 42 KDa indicates that Biotinylated compound binds to RPN13and the band disappears when the cell lysate is pretreated with the samenon-biotinylated compound.

Methods

Chemistry:

All reagents and solvents were obtained from Aldrich. Anhydrous solventswere used as received. Reaction progress was monitored with analyticalthin-layer chromatography (TLC) plates carried out on 0.25 mm MerckF-254 silica gel glass plates. Visualization was achieved using UVillumination. ¹H NMR spectra were obtained at 400 MHz on a Bruker Avancespectrometer and are reported in parts per million downfield relative totetramethysilane (TMS). EI-MS profiles were obtained using a BrukerEsquire 3000 plus. Crude compounds were purified by semi-preparativereversed phase HPLC using a Water Delta Prep 4000 system with aPhenomenex column C18 (30×4 cm, 300 A, 15 μm spherical particle sizecolumn).

Construction of RPN13 Expression Plasmid:

The human RPN13 cDNA was obtained from OriGene™ (ARDM1, NM_175563) andsubcloned into pET28a(+) vector (Novagen™) at the BamH1 and Xho1restriction sites after PCR using forward and reverse primers to yieldRPN13-pet28a (+) which expresses a recombinant RPN13 with ahexahistidine-tag at both the N and C terminus. RPN13 is a 42 kDaprotein however, because of the additional sequences on the pET28a (+)vector, inclusive of the hexahistidine tags at both ends, the overallmolecular weight of recombinant tagged RPN13 was ˜49 kDa.

Recombinant RPN13 Preparation:

Human RPN13 full length, RPN13 (1-150), RPN13 (253-407), and His-taggedUCH37 were prepared as described.³¹

Bacterial Strains and Expression of RPN13:

For protein expression, briefly, RPN13-pET28a (+) was transformed intoE. coli 2 (DE3) cells (Rosetta 2 Cells, Novagen™). Single colonies oftransformed cells were picked, suspended into 10 mL of Superbroth andgrown aerobically at 250 rpm, 37° C. overnight in 15 mL round bottomtubes (BD Falcon). The next day, 1 mL of the culture was collected andsuspended in a new 15 mL round-bottom tube containing 5 mL of freshSuperbroth. The culture was then grown again aerobically at 250 rpm,370C and monitored for log phase. When the bacteria reached the middleof log phase (OD600=˜0.6), 1 mM of isopropylthio-β-D-galactoside (IPTG)was added to induce RPN13 protein expression. In general, it took 2 hrfor O.D600 to reach ˜0.6. The culture was induced for 3 hr, and 500 μLof the culture was centrifuged. The cells collected were suspended in100 μL of PBS and 10 μL of the suspension was then collected andanalyzed by Tris-Glycine SDS-PAGE.

Plasmid Overexpression Studies:

RPN13, RPN10 plasmids were obtained from Origene™. UCH37 (plasmid 19415)and RAD23/HHR23B (plasmid 13054) plasmids were obtained from Addgene.pCMV-FL plasmid was a kind gift from Dr. David Piwnica Worms. 293TTcells were transfected with the above plasmids with TransIT-2020transfection reagent for 48 hr per the manufacturer's recommendation(Mirus Bio LLC). Cells were lysed with MPER lysis buffer and subjectedto Western blot analysis for biotin recognition studies as described inthe biotin labeling assay section.

Cell Culture:

All cell lines were obtained from American Type Culture Collection(Manassas, Va.) except 293TT cells from Dr. C. Buck (NCI, NIH), and werecultured in specified medium supplemented with 10% fetal bovine serum,100 IU/mL penicillin, and 100 μg/mL streptomycin at 5% CO2. 293, 293 TT,HeLa, SiHa, ME180, HT3 and C33A were grown in DMEM, CaSki, ES2,NCI-H929, MM.1S, RPMI-8226, U266, ANBL6 and Bortezomib resistant linesin RPMI-1640, and SKOV3 and HCT116 cell lines in McCoys 5A Medium. 293TTcells were transfected with RPN13, RPN10 (Origene), UCH37, and HHR23B(Addgene) expression vectors using TransIT-2020 transfection reagent perMirus Bio's instructions.

Cell Viability Assays:

Cell viability was assayed using CellTiter 960 AQueous One SolutionReagent (Promega, Madison, Wis.). Cells seeded at the concentration of1,000 cells/well for HeLa, 5,000 cells/well for ES2 and HCT116 and10,000-20,000 cells/well for other cell lines in 100 μL medium in96-well plate were treated with RA series compounds at specifiedconcentrations. After the indicated periods, cells were incubatedaccording to the manufacturer's protocol with the MTS labeling mixturefor 1-2 hr and absorbance at 490 nm measured using a Benchmark Plusmicroplate spectrophotometer (BIO-RAD).

Quantitative PCR to Measure mRNA Levels.

Quantitative PCR was performed per the manufacturer's instructions(Applied Biosystems), according to the Livak method and normalized toreference gene GAPDH.s described elsewhere.^(3,6) Total RNA was isolatedfrom cells using the RNeasy mini kit (Qiagen). Extracted RNA wasnormalized for concentration and reverse transcribed using iScript cDNAsynthesis kit (Bio-Rad). CHOP-10 expression levels were measured byTaqman gene expression assays with Taqman gene expression master mix(Applied Biosystems) and run with the standard thermal cycling protocol.Spliced XBP1 mRNA was assayed with SsoFast EvaGreen Supermix (Bio-rad)following the protocol for the iCycler System. Calculations were doneaccording to the Livak method and normalized to reference gene GAPDH.Each condition was replicated three times; each sample was run intriplicate.

Flow Cytometry.

An annexin V-PE apoptosis detection kit I (BD PharMingen) was usedaccording to the manufacturer's protocol. Cell surface HSP90 was stainedwith anti-HSP90 mAb (Stressgen), followed by PE-conjugated anti-mouseIgG1. The data were acquired with a FACSCalibur and analyzed usingCellQuest software.

Determination of Apoptotic Cells by Flow Cytometry:

Induction of apoptosis was determined using Annexin V-PE ApoptosisDetection Kit I (BD Pharmingen, San Diego, Calif.) according to themanufacturer's protocol. Briefly, 1×10⁵ cells were re-suspended inbinding buffer, 5 μL of Annexin V-PE and 5 μL of 7-AAD were then addedinto the cells, which were then incubated at room temperature for 15 minand analyzed by flow cytometry as above.

Antibodies and Western Blot Analysis.

Total cellular protein (10-20 μg) from each sample was subjected toSDS-PAGE, transferred to PVDF membranes and analyzed by Western blot.Antibodies for Western blot analysis were obtained by the followingcommercial sources: anti-ubiquitin, anti-p53 (FL393), anti-p21(WAF1),anti-hDLG-1(2D11), anti-Puma, Bak, Bax (Cell Signaling Technology,Danvers, Mass.), anti-IκB-α (C-15), anti-ATF-4 (SC-200) (Santa CruzBiotechnology, Santa Cruz, Calif.), anti-PARP (BD Pharmingen, San Diego,Calif.), anti HSP90 (Stressgen Corp, Victoria BC, Canada), anti-tubulin(Sigma, St. Louis, Mo.), anti-Actin, anti-ADRM1 (anti-RPN13), anti-RPN2(anti-PSMD1) (Sigma-Aldrich), anti-RPN10, UCH37 (Cell-signaling),anti-RPN1 (proteasome 19S subunit anti-S2) (Thermo Scientific),streptavidin Dyna beads (Invitrogen), HRP-streptavidin (Invitrogen),peroxidase-linked anti-mouse Immunoglobulin G and anti-rabbit IgG (GEHealthcare UK Ltd, UK) and utilized at the concentration recommended bythe manufacturer.

Enzymatic Assays.

Reaction mixtures contained 500 nM of the purified 19S RP (R&D Systems)in 19S enzyme assay buffer and 1 μM Ub-AMC. Release of free AMC by theenzyme reaction was determined by measuring its fluorescence at 380 nm(excitation) and 440 nm (emission). The 20S proteolytic activity assayswere performed as described elsewhere (Bazzaro et al., 2011).

Biotin Labeling Assay:

Human cells (5×10⁶ HeLa or 293TT cells) were lysed using MPER (Pierce)lysis buffer (500 μL). RPN13 or HPV L2 transformed bacterial cells (500μL of culture) were centrifuged to harvest the cells which werere-suspended in 500 μL BPER (Pierce). Cell lysates were centrifuged at10,000 rpm briefly (2 min) at 4° C. to remove cell debris. Lysatesupernatant (200 μL) was pre cleared with streptavidin dyna beads (20μL) for 1 h at 4° C. to remove non-specific biotin binding and incubatedwith compounds (indicated concentrations or 20 μM) at 4° C. for 1 hr. Anequal amount of each sample (20 μL of lysate) was mixed with an equalvolume of Laemmli sample buffer (20 μL) (BioRad) and was boiled for 5min. The proteins were separated using 4-15% Bio-Rad Mini-PROTEANSDS-PAGE gel (1 hr at 100 V) and transferred to the membrane overnightat 4° C. (24 V). Membrane was blocked with 5% BSA in PBST for 1 hr atroom temperature and washed for 20 min (3×PBST). Then the membrane wasprobed with HRP-streptavidin (1:10,000 in PBST) for 1 hr at roomtemperature and washed for 30 min (3×PBST) and developed using HyGLOchemiluminescent detection reagent (Denville) for biotin recognition.For the purified enzyme assay, 19S purified proteasome (R&D Systems,Cat. No. E-366) (500 ng) in 19S buffer (20 mM HEPES, 20 mM NaCl, 1 mMDTT, 15% Glycerol) was incubated with compounds (20 μM) for 1 hr at roomtemperature and mixed with an equal volume of Laemmli sample buffer (20μL) (BioRad) and was boiled for 5 min. Each sample was loaded onto geland followed the western blot protocol mentioned above.

Drug Combination Index Assay:

HeLa cells seeded at 5,000 cells/well in 100 μL medium in 96-well platewere treated with RA190 (0-300 nM) and/or bortezomib (0-30 nM) alone orin combination. After 48 hr, cells were incubated according to themanufacturer's protocol with the MTS labeling mixture (CellTiter 960AQueous One Solution Reagent, Promega, Madison, Wis.) for 1 hr andabsorbance at 490 nm measured using a Benchmark Plus microplatespectrophotometer (BIO-RAD). The combination indices and a normalizedisobologram were calculated using Compusyn software (www.combosyn.com).

Cell Surface HSP90 Staining:

Cell surface HSP90 was detected by staining with purified anti-HSP90 mAb(Stressgen), followed by PE-conjugated anti-mouse IgG1. The data wereacquired with a FACSCalibur and analyzed using CellQuest software.

Glycerol-Gradient Purification:

293 cells (20×10⁶) were lysed in MPER lysis buffer (2 mL) usingmanufacturer recommended protocol (Pierce). Lysate was precleared at9,000 rpm using table top centrifuge at 4° C. for 2 min. Supernatantwere collected and 1 mL of lysate was incubated with RA190 (20 μM) at 4°C. for 1.5 hr and another 1 mL was incubated with DMSO as a control.Both samples were loaded separately on top of a 10-40% (v/v) glycerolgradient (13 mL) and centrifuged at 27,000 rpm for 15 hr at 4° C. in aBeckman SW 40 Ti rotor. Fractions (0.8 mL) were collected from the topof the gradient and analyzed for proteasomal proteins by Western blotanalysis.

19S Ub-AMC Assay:

Proteasomes retain high levels of ubiquitin-AMC (Ub-AMC) hydrolyzingactivity, which is presumably dependent on 19S RP subunits USP14 andUCH37. To identify whether RA190 has any influence on these enzymes, weperformed an in vitro assay with 19S RP. Reaction mixtures contained 500nM of the purified 19S RP in 19S enzyme assay buffer and 1 μM Ub-AMC.After incubation of the mixtures with corresponding compounds forvarious concentrations and time periods at 37° C., release of free AMCby the enzyme reaction was determined by measuring its fluorescence at380 nm (excitation) and 440 nm (emission). The graphs shown arerepresentative of at least two independent determinations and each datapoint is the mean of ±SD of triplicate determinations.

20S Proteolytic Activity Assay:

Purified 20S proteasomes were treated for 30 min with or withoutcompounds (1 μM) or Bortezomib (1 μM) prior to the addition of thespecific fluorogenic substrate for chymotrypsin (Suc-LLVY-AMC), tryptic(Boc-LRR-AMC) or PGPH (Ac-YVAD-AMC) hydrolytic proteasome capacities.Release of free AMC by the enzyme reaction was determined by measuringits fluorescence at 380 nm (excitation) and 440 nm (emission). Thegraphs shown are representative of at least three independentdeterminations and each data point is the mean of ±SD of triplicatedeterminations.

NMR spectroscopy:

NMR spectra were acquired at 25° C. or 10° C. on Bruker NMRspectrometers operating at 850 or 900 MHz and equipped withcryogenically cooled probes. Processing was performed in NMRPipe¹³ andthe resulting spectra visualized with XEASY.³ Protein concentrationswere calculated by extinction coefficients based on amino acidcomposition and absorbance at 280 nm for protein dissolved in 6 Mguanidine-HCl. Buffer A (20 mM NaPO4, pH 6.5, 50 mM NaCl, 2 mM DTT, 0.1%NaN3, and 5% D20) was used for all NMR samples except those containingRA190, which were performed in Buffer B (Buffer A with no DTT present).10-fold molar excess RA190 (5 mM stock in DMSO) was incubated with RPN13protein at 4° C. overnight and unreacted RA190 removed by dialysis.

LC High Resolution Mass Spectrometry:

RPN13 Pru domain and RA190-exposed RPN13, RPN13 Pru domain, RPN13 PruC60,80,121A, and RPN13 Pru C88A at protein concentrations of 1 μg/μLwere treated with formic acid and injected onto a Nano2D-LC HPLC system(Eksigent, Dublin, Calif.) equipped with an Agilent Zorbax 300SB C8column (3 mm ID, 10 cm length, 3.5 μm particle size). The samples weresubjected to 10 min of incubation in 98% H2O:2% CH3CN, a 20 min lineargradient at 10 μL/min flow rate to 75% H2O:25% CH3CN followed by a 12min linear gradient to 5% H2O:95% CH3CN and then a 2 min hold. Analysiswas conducted by positive ionization electrospray with an LTQ-OrbitrapVelos instrument (Thermo Scientific, Waltham, Mass.) and adducts werequantified utilizing the Orbitrap detector with a resolution of 100,000at a scan range of 400-2000 m/z.

RPN13 Pru˜RA190 Complex Calculation:

The RPN13 Pru˜RA190 complexes were generated by using HADDOCK 2.1 (HighAmbiguity Driven protein-protein DOCKing)¹⁵ in combination with CNS(Brunger et al., 1998). A homology model of RPN13 Pru domain wasgenerated by Schrödinger based on the atomic coordinates of murine RPN13Pru domain (PDB entry 2R2Y). ³⁴ Four distance restraints were definedbetween C88 Sγ of RPN13 and four atoms of RA190 to recapitulate thesulfur-carbon bond (FIG. 12D and Table 4); HADDOCK requires two distinctmolecules for docking. The ratio of peak intensity values (Δ) wereplotted for each RPN13 backbone (FIG. 4A) and side chain (FIG. 4B) amidegroup according to Equation 1, in which/represents peak intensity and0.588 is a scaling factor derived by setting the randomly coiled N and Cterminal ends of RPN13 Pru domain as unaffected by RA190.

Δ=1−(0.588x/ _(RPN13 Pru-RA190)//_(RPN13 Pru))  Equation 1:

TABLE 4 Unambiguous distance restraints used in HADDOCK to define RA190attachment to RPN13 C88 Sγ. RPN 13 Pru RA190 Distance (in Angstroms)C88Sγ CAI 1.829 C88Sγ HAI 2.384 C88Sγ CAZ 2.854 C88Sγ CBC 2.735

RPN13 Pru residues with Δ values greater than one standard deviationvalue above average were defined as “active” and their neighbors as“passive” provided they have >40% accessibility. Ambiguous InteractionRestraints (AIRs) were imposed to restrict hRPN13 Pru active residues tobe within 2.0 Å of any RA190 atom (Dominguez et al., 2003). 1000structures were subjected to rigid-body energy minimization and the 250lowest energy structures chosen for semi-flexible simulated annealing intorsion angle space followed by refinement in explicit water. Duringsemi-flexible simulated annealing, atoms at the interface were allowedto move, but constrained by the AIRs and unambiguous distanceconstraints defining the sulfur-carbon bond. After water refinement, theRMSD of RA190 in the resulting 250 structures was 3.81±0.60 Å and thesewere sorted into four clusters by using a 1.5 Å cut-off criterion. Thelowest energy structure of each cluster was energy minimized bySchrödinger after the explicit introduction of a covalent bond betweenRPN13 C88 Sy and RA190 reacted carbon.

TABLE 5 Statistics for human RPN13~RA190 structures sorted into fourclusters by using a 1.5 Å cut-off criterion. Number Cluster of HaddockNo. Structures Score RMSD E_(inter) E_(vdw) E_(elec) BSA 1 82 61.18 ±8.21  0.85 ± 0.33 183.35 ± 16.12 69.25 ± 4.30 −10.82 ± 4.37  662.04 ±36.10 2 30 69.43 ± 12.85 0.86 ± 0.33 192.31 ± 24.71 76.97 ± 3.86 −6.70 ±4.46 540.38 ± 28.91 3 29 76.45 ± 7.16  0.95 ± 0.31 198.22 ± 12.11 81.17± 2.32 −3.64 ± 2.73 470.19 ± 34.17 4 67 81.62 ± 7.34  1.07 ± 0.31 211.25± 14.20 80.31 ± 2.20 −2.35 ± 2.06 482.75 ± 30.00

E_(inter), intermolecular binding energy; E_(vdw), van der Waals energy;E_(elec), electrostatic energy; BSA, buried surface area; RMSD, rootmean square deviation for backbone atoms to the cluster's averagestructure.

Cell Culture Assay for Luciferase:

Sub-confluent cultures of HeLa cells were transfected with 4UbFL or FLplasmids using Lipofectamine 2000 reagent (Life Technologies, Carlsbad,Calif.). HeLa cells were seeded at 20,000 cells/well in a 96-well plate48 hr post transfection and incubated with compounds or vehicle (DMSO)at the doses or time indicated for individual experiments. Luciferaseactivity in cell lysate was determined with a luciferase assay kit(Promega, Madison, Wis.) according to the manufacturer's instructions.Bioluminescence was measured by using a Glomax Multidetection system(Promega, Madison, Wis., USA).

Animal Studies.

All animal experiments were performed in accordance with protocolsapproved by the Animal Care and Use Committee of Johns HopkinsUniversity. Nude, C57BL/6, Balb/c female mice were purchased from theNational Cancer Institute (NCI) and NOG (NOD/Shi-scid/IL-2Rγnull) micewere bred in-house. DNA delivery was via a helium-driven gun (Bio-Rad)as described previously⁴⁰ or electroporation (ElectroSquarePorator 833,BTX-2 Needle array 5 mm gap, Harvard apparatus) delivered as eightpulses at 106 V for 20 ms with 200 ms intervals. Bioluminescence imageswere acquired for 10 min with a Xenogen IVIS 200.²⁵ C57BL/6 mice werechallenged subcutaneously with 5×10⁴ TC-1 cells/mice and E7-specificCD8+ T cell response and tumor size were measured as describedpreviously (Trimble et al., 2003). Nude mice were inoculated with 1×10⁶EΩ-luciferase cells i.p. in 100 μl PBS. At day 3, mice were imaged forbasal level luciferase activity. NOG mice (five per group) wereinoculated with 1×10⁶ NCI-H929-GFP-luc cells i.v., and imaged for basalluciferase activity after 4 weeks.

Pharmacokinetic Measurements:

Mice were humanely euthanized, and plasma was harvested as a function oftime after administration of RA190. Tissue was harvested at 48 hr aftersingle-dose intraperitoneal administration in non-tumor bearing mice.Blood was collected by cardiac puncture under anesthesia intoheparinized syringes and centrifuged to obtain plasma. Tissues wererapidly dissected and snap frozen in liquid nitrogen. Samples werestored frozen at −70° C. until analysis. RA190 was extracted from mouseplasma using acetonitrile containing temazepam (100 ng/mL). Tissuehomogenates were prepared at a concentration of 200 mg/mL in PBS andfurther diluted 1:10 in mouse plasma before extraction usingacetonitrile containing temazepam (100 ng/mL). Separation was achievedon Waters X-Terra™ C₁₈ (50 mm×2.1 mm i.d., 3 μm) at room temperatureusing isocratic elution with acetonitrile/water mobile phase (70:30,v/v) containing 0.1% formic acid at a flow rate of 0.2 mL/min. Detectionwas performed using electrospray MS/MS operating in negative mode bymonitoring the ion transitions from m/z 561.0→119.8 (RA190) and m/z301.2→225.0 (temazepam). Samples were quantitated over the assay rangeof 10 to 1000 ng/mL or 0.6 to 60 μg/g. Mean plasma concentrations ateach sampling point were calculated for RA190. Pharmacokinetic variableswere calculated from mean RA190 concentration-time data usingnoncompartmental methods as implemented in WinNonlin Professionalversion 5.3 (Pharsight Corp., Mountain View, Calif.). Cmax and Tmax werethe observed values from the mean data. The AUC_(last) was calculatedusing the log-linear trapezoidal rule to the end of sample collection(AUC_(last)) and extrapolated to infinity (AUC_(0-∞)) by dividing thelast quantifiable concentration by the terminal disposition rateconstant (λz), which was determined from the slope of the terminal phaseof the concentration-time profile. The half-life (T_(1/2)) wasdetermined by dividing 0.693 by λ_(z). Relative bioavailability wascalculated as follows:

${{Relative}\mspace{14mu} {Bioavailability}\mspace{14mu} (\%)} = {\left( \frac{{AUC}_{{0 - \infty},{p.o.}}}{{AUC}_{{0 - \infty},{i.p.}}} \right)*\left( \frac{{Dose}_{i.p}}{{Dose}_{p.o.}} \right)}$

Pharmacokinetic Parameters were Summarized Using Descriptive Statistics.

Complete blood count and blood chemistry analyses: After carbon dioxideeuthanasia, 500 to 600 μL blood was collected from each mouse byintracardiac aspiration with a 25-gauge needle and 1-mL syringe. Bloodwas placed in a 600 μL centrifuge tube coated with lithium heparin toprevent clotting. A complete blood count was performed with an automatedhemocytometer (Hemavet HV950FS, Drew Scientific, Oxford, Conn.). Theremaining blood was centrifuged and the plasma drawn off and analyzedwith an automated clinical chemistry analyzer (VeTACE, Alfa Wassermann,West Caldwell, N.J.).

In vivo DNA delivery: Gene gun particle-mediated DNA delivery wasperformed using a helium-driven gene gun (Bio-Rad, Hercules, Calif.) asdescribed previously (Trimble et al., 2003). For electroporation, apatch of the mouse leg was shaved and 10 μg 4UbFL or FL DNA plasmid in20 μL of PBS was injected into the quadriceps femoralis muscle followedimmediately by injection of the 2 Needle Array to 5 mm depthencompassing the injection site and square wave electroporation(ElectroSquarePorator 833, BTX-2 Needle array 5 mm gap, Harvardapparatus) delivered as eight pulses at 106 V for 20 ms with 200 msintervals.

In Vivo Mouse Imaging:

Two hours after gene gun delivery of the plasmid or one day postelectroporation, mice were injected i.p. with 100 uL of luciferin (3mg/mL) and anesthetized with isoflurane and optical imaging wasperformed for basal level luciferase expression. Images were acquiredfor 10 min with a Xenogen IVIS 200 (Caliper, Hopkinton, Mass.). Equallysized areas were analyzed using Living Image 2.20 software. Mice wereagain imaged at 4 hr and 24 hr post treatment.

In Vivo Tumor Treatment:

For TC-1 tumors, C57BL/6 mice (8/group) were challenged subcutaneouslywith 5×104 TC-1 cells/mice. Tumors grew for approximately 7 days untilthey were palpable, whereupon the mice were weighed and treatment wasinitiated. Tumor size was measured with a digital caliper and calculatedbased on the formula: [largest diameter×(perpendicular diameter)2]π/6.Nude mice (8 per group) were inoculated with 1×106 EΩ-Luciferase cellsi.p. in 100 μL PBS. At day 3 mice were imaged for basal levelluminescence expression. Mice were divided into two groups and treateddaily i.p. with RA190 (10 mg/kg) or vehicle, and imaged again on day 7and day 14. NOG mice (5 per group) were inoculated with 1×106NCI-H929-GFP-Luc cells i.v., and after 4 weeks, mice were imaged fortheir luciferase activity and divided into two groups. Mice were treatedi.p. with RA190 (20 mg/kg) or vehicle as indicated, and imaged again atthe end of the treatment for their luciferase activity.

E7-Specific CD8+ T Cell Response:

Splenocytes were prepared and stimulated with HPV16 E7aa49-57 peptide (1μg/mL) at the presence of GolgiPlug (BD Pharmingen) overnight at 37° C.The cells were first stained with PE-conjugated anti-mouse CD 8α (BDPharmingen), then permeabilized, fixed and intracellularly stained withFITC-conjugated anti-mouse IFN-γ (BD Pharmingen). The data were acquiredwith FACS Calibur and analyzed with CellQuest software.

Statistical Analysis:

Results are reported as mean±standard deviation (s.d.). Statisticalsignificance of differences was assessed by two-tailed Student's t usingPrism (V.5 Graphpad, San Diego, Calif.) and Excel. The level ofsignificance was set at p≤0.05.

Preparation of RA Series Candidate Inhibitors:

Reagents and conditions: (a) AcOH, dry HCl gas, room temperature,overnight (b) Boc or Fmoc protected amino acid, HOBt, HBTU, DMF, DIPEA,room temperature, 3 hr (c) 20% piperidine in DMF (d) 4 M HCl in dioxane.

Compounds in Tables 6 and 7 were synthesized utilizing classicalsolution phase reactions as seen in FIG. 31. In FIG. 31, substitutedbenzaldehyde (s) II (2.0 mmol) was added to a suspension of 4-piperidonehydrochloride monohydrate I (1.0 mmol) in glacial acetic acid (15 mL).Dry hydrogen chloride gas was passed through this mixture for 0.5 hrduring which time a clear solution was obtained. After standing at roomtemperature for 24 hr, the precipitate (AcOH salt of benzylidinepiperidone) Ill was collected and dried under the vacuum. Targetcompound (s) IV were synthesized by reacting corresponding compound (s)III and corresponding amino acids in the presence of peptide couplingagents. All the compounds were purified by HPLC and characterized by MSand NMR. For the deprotection of Boc functionality, 4M HCl in dioxanesolution was used.

The general chemical structure of effective molecules is the following:

The pair of A groups represents a phenyl substituted with 1-5substituents selected from the group consisting of R1, OR1, NR1R2,S(O)_(q)R1, SO₂NR1R2, NR1SO₂R2, C(O)R1, C(O)OR1, C(O)NR1R2, NR1C(O)R2,NR1C(O)OR2, halogen, CF₃, OCF₃, cyano, and nitro where R1 and R2represent hydrogen, nitro, hydroxyl, carboxy, amino, halogen, and cyanoor C₁-C₁₄ linear or branched alkyl groups, that are optionallysubstituted with 1-3 substituents selected from the group consisting ofC₁-C₁₄ linear or branched alkyl, up to perhalo substituted C₁-C₁₅ linearor branched alkyl, C₁-C₁₄ alkoxy, hydrogen, nitro, hydroxyl, carboxy,amino, C₁-C₁₄ alkylamino, C₁-C₁₄ dialkylamino, halogen, and cyano.

The phenyl ring can also be replaced with a naphtha group optionallyhaving 1-5 substituents chosen from the same list as the phenylsubstituents or a 5 or 6 membered monocyclic heteroaryl group, having1-3 heteroatoms selected from the group consisting of O, N, and S,optionally substituted with 1-3 substituents chosen from the same listas the phenyl substituents or an 8 to 10 membered bicyclic heteroallylgroup containing 1-3 heteroatoms selected from the group consisting ofO, N, and S; and the second ring is fused to the first ring using 3 to 4carbon atoms, and the bicyclic hetero aryl group is optionallysubstituted with 1-3 substituents selected from the group consisting ofR1, OR1, NR1R2, S(O)_(q)R1, SO₂NR1R2, NR1SO₂R2, C(O)R1, C(O)OR1,C(O)NR1R2, NR1C(O)R2, NR1C(O)OR2, CF₃, and OCF₃.

X is OR1, NP, wherein P is selected from the group consisting of H, R1,C(O)R1, C(O)OR1, C(O)NR1R2, S—N(R1)COOR1, and S—N(R1). Y, NR1 or CR1R2.Y is selected from the group consisting of O, S, NR1, CR1R2. Z isselected from the group consisting of hydrogen; C₁ to C₁₄ linear,branched, or cyclic alkyls; phenyl; benzyl, 1-5 substituted benzyl, C₁to C3 alkyl-phenyl, wherein the alkyl moiety is optionally substitutedwith halogen up to perhalo; up to perhalo substituted C₁ to C14 linearor branched alkyls; —(CH₂)_(q)—K, where K is a 5 or 6 memberedmonocyclic heterocyclic ring, containing 1 to 4 atoms selected fromoxygen, nitrogen and sulfur, which is saturated, partially saturated, oraromatic, or an 8 to 10 membered bicyclic heteroaryl having 1-4heteroatoms selected from the group consisting of O, N and S, whereinsaid alkyl moiety is optionally substituted with halogen up to perhalo,and wherein the variable q is an integer ranging from 0 to 4

An embodiment with phenyl groups in place of each A pair is as follows:

To understand the possible variations of the R1 and R2 chains, thefollowing examples are provided:

The structure above represents an R1 which is a linear alkyl chain of nrepeating units.

Above, R1 is a branched alkyl chain.

Above, R1 is a branched alkyl chain with additional substituents.

Above, R1 is a branched perhalo substituted alkyl chain.

Above, R1 is an alkoxy chain (with oxygen at any position)

Above, R1 is hydroxy.

Above, R1 is Carboxy and COR1.

Above, R1 is C₁-C₁₄ Alkyl Amino

Above, R1 is C₁-C₁₄ Di Alkyl amino

Above, R1 is any halogens (Cl, F, Br, I)

Above, R1 is Cyano.

Above, R1 is Nitro.

Above, R1 is CF₃(perfluoro).

Above, R1 is SO2NR1R2

Above, R1 is NR1SO2R2

In this embodiment “D” Can be Oxygen, Nitrogen, Sulfur located anywherein C₁-C₁₄ carbon chain

Here R1 is S(O)qR1 (Wherein the variable q is an integer selected from0,1,2,3, or 4).

Here R1 is CONR1R2

Here R1 is NR1COR2

Here R1 is NR1COOR2.

Chemical Structures of RA Compounds.

Chalcones are Michael acceptors and thus their activity is modulated byelectron withdrawing/donating character of substituents at the ortho andpara positions of the aromatic ring. Therefore in the present study thechalcone functionality has been attached to a piperidone nucleus. Wegenerated a new bis-benzylidine piperidone scaffold that incorporatestwo Michael enones in a single molecule and introduced differentsubstituents in the aromatic ring to modulate the acceptor character ofthe enone system of chalcones. Using this strategy, a series ofcompounds was synthesized by incorporating halogens at ortho and parapositions and different amino acids at the amine functionality of4-piperidone. Since our previous work suggested the importance ofphenylalanine at the amine functionality of 4-piperidone for proteasomeinhibitory activity,⁵ we derived a majority of RA compounds byincorporating phenylalanine and/or substituted phenylalanine at the sameposition along with the halogen substituents in the aromatic rings. Toovercome the poor solubility and pharmacokinetics of our previousgeneration molecules, we employed an amide in lieu of a urea linkagebetween amino acids and 4-piperidone. We synthesized RA166 and RA201with halogens chlorine and fluorine at the ortho position of aromaticrings and phenylalanine attached to the 4-piperidone. Our priormolecular modeling studies suggested the importance of having twochlorine atoms on one phenyl moiety and thus we synthesized RA190,RA190Ac which possess differences in the phenylalanine and amideconjugation as opposed to the urea conjugation of our first generationmolecule RA1.⁵ To gain insight into the importance of phenylalanine, wesynthesized compounds RA196 and RA213 in which histidine and tyrosineare substituted for phenylalanine, and RA181, which has no substituent.Structures for RA190B and RA190ME are also provided.

Rationale for Designing Molecules:

As electrophilic agents, Michael acceptors may form covalent bonds tonucleophilic sites of proteins of biological organisms. In general,Michael acceptors (these are also called α, β-unsaturated esters,unsaturated ketones, etc.) are alkenes attached to electron-withdrawinggroups (esters, ketones, nitriles, etc.). The electrophilic reactivityof Michael acceptors is an important determinant of their biologicalactivity. In our bis-benzylidine piperidone pharmacophore, a typicalMichael acceptor (in which, only one carbonyl moiety shares with twoalkenes) is attached to two substituted aromatic rings at theirβ-carbon. Because of this arrangements aromatic ring electrons canresonate with carbonyl moiety in the Michael acceptor and ringsubstituents play an important role in acceptor activity.

We hypothesized that the electron withdrawing effects, number ofsubstituents and their positions on the aromatic ring would facilitatethe nucleophilic addition on the Michael acceptor in the binding site onRPN13. In our previous work we demonstrated that amino acid substituentsat the secondary amine of the piperidone moiety showed greater efficacytowards the proteasome inhibition as compared to the free amine. Keepingall these criteria in view we synthesized several analogs ofbis-benzylidine piperidone moiety through rational drug design.

We used Chloro and Nitro functional groups as substituent's on thearomatic ring due to their greater electron withdrawing effect at ortho(or) para (or) meta (or) both positions along with different amino acidlinkages on bis-benzylidine piperidone moiety. Amino acids were chosenbased in their physical characteristics like very hydrophobic(phenylalanine), strongly basic and hydrophilic (lysine), basic andhydrophilic (histidine) and amphiphilic (glycine). Commerciallyavailable Fmoc and Boc protected amino acids were used in the synthesis,and they were evaluated for their antiproliferatory activity againstcancer cells. Molecules with Fmoc groups showed some impact on cancercell proliferation but they were not soluble and potent because of thelarge size of the Fmoc group and possible steric hindrance. Hence, wedeprotected the Fmoc group and the free amine analogs were tested fortheir efficacy. These new analogs with free amine showed greater antiproliferative efficacy against cancer cells but lack the solubility. Toovercome this limitation, next we synthesized HCl salts ofbis-benzylidine piperidones and they were more potent and solved thesolubility issues associated with free amines.

Among all the analogs, Phenylalanine (RA183 and RA190) and lysine(RA195) containing molecules showed higher potency towardantiproliferative activity and accumulated Poly-ubiquitinated proteinsin cancer cells. Next we synthesized another set of compounds byconverting free amine functionality to its Acetate (Ac), Acrolyl (Acr)and Benzoyl (Bn) analogs as these units are part of many anticancerdrugs because of their smaller size and feasibility to form intermolecular hydrogen bonds with proteins. Even though these new analogswere highly potent, HCl salts were chosen for further studies because oftheir better solubility.

Many cancer cells were highly sensitive to RA190, hence we synthesizedother analogs of RA190 which includes RA190P (amine functionality isconverted to picolinic acid), RA190D (D-phenylalanine was used insteadof L-Phenylalanine). Neither of these compounds was as good as RA190. Inthe RA195 molecule two amino functionalities of lysine can provide moreflexibility to make better analogs. Hence, we synthesized three RA195analogs with different functional groups at δ-amine which includesRA195F (Fmoc HCl salt), RA195Ac (Acetate HCl salt) and RA195Bn (BenzoylHCl salt). FIG. 25 suggests that Acetate and Benzoyl versions of RA195are more potent than the parent molecule RA195.

Rationale for RA190-Folate and RA190-Biotin:

Targeted delivery of high doses of chemotherapeutics using ligandshighly needed for cancer cell survival may be an attractive alternativeapproach for the successful treatment of cancer. Such important ligandsinvestigated for targeted drug delivery in recent years are folic acidand biotin.

Many cancer cells have a high requirement for folic acid and overexpressthe folic acid receptor. This finding has led to the development ofanti-cancer drugs that target the folic acid receptor. Folate isimportant for cells and tissues that rapidly divide. Cancer cells dividerapidly, and drugs that interfere with folate metabolism are used totreat cancer. The anti-folate methotrexate is a drug often used to treatcancer because it inhibits the production of the active tetrahydrofolate(THF), which is required to synthesize nucleic acids, from the inactivedihydrofolate (DHF). However, methotrexate can be toxic, producing sideeffects, such as inflammation in the digestive tract that make itdifficult to eat normally. We use folate drug delivery approach forRA190 to target cancer cells. In this method we introduce folic acidinto RA190 directly or through a spacer to make folate-RA190 (RA190-F)synthetically. RA190-Folate should have more specificity towards cancercells as these cells are highly dependent on Folic acid and RA190-Folateand its analogs can compete with Folic acid.

Biotin is essential for cell growth, the production of fatty acids, themetabolism of fats and amino acids, and growth and development. Humanscan't synthesize biotin and it is generally available from exogenousdietary sources and from intestinal bacteria. The high metabolisms ofcancer cells make them depend on vitamins such as biotin (vitamin B7).Biotin levels were found to be significantly higher in many cancersespecially colon and ovarian cancer tissues compared to normal tissue.At the same time these tumor cells over express biotin receptors alongwith folate receptors. Several research groups are pursuing biotinylatedpro-drugs that target biotin receptors. For example, biotinylatedconjugates of camptothecin have been shown to be more cytotoxic andinduce apoptosis by activation of the caspase-dependent cell deathsignaling pathway and were effective against multidrug resistant ovariancarcinoma cells. High availability and flexible syntheses makesbiotinylated pro-drugs open up a new avenue in targeted drug deliveryfor overcoming resistance to chemotherapy.

Following are examples of biotin and folate versions of the inventivemolecules. As an overall framework the molecules can be envisioned asbeing based on the following generalized structure where the “ball”

represents Biotin, Folic acid, Methotrexate (MTX), or Pteroic acid.

A number of molecules constructed on the same basic structure follow:

TABLE 6 Molecular formulae

D₁ D₂ D₃ E RA166 Cl H H

RA181 H H H

RA190 H Cl Cl

RA190Ac H Cl Cl

RA196 H Cl Cl

RA201 F H H

RA213 H Cl Cl

TABLE 7 Additional Molecular Formulae:

RA181F

RA182

RA183

RA183Acr

RA183Biotin (RA183B)

RA184

RA186

RA190F

RA190Acr

RA190P

RA190D

RA190ME

RA190O

RA190R

RA190Biotin (RA190B)

RA191

RA193

RA194

RA195

RA195Ac

RA195Bn

RA195Biotin (RA195B)

RA 195B (a)

RA190- spacer-B

RA183- spacer-B

RA195- spacer-B

RA195- spacer-B(a)

RA190F

RA183F

RA195F

RA195F(a)

RA190- spacer-F

RA183- spacer-F

RA190- spacer-F

RA195- spacer-F(a)

RA190-TFA

RA190-PIP

TABLE 8 Chemical structures of a few related analogs of bis-BenzylidinePiperdone

C1

C2

C3

C4

C5

C6

C7

Analytical Data for RA compounds:

3,5-bis(2-chlorobenzylidene)-1-(S-2-amino-3-phenyl)-piperidin-4-one-HClsalt (RA166): ¹H NMR (DMSO-d6): δ 7.90 (d, 2H, J=8 Hz), 7.11-7.53 (m,11H), 6.78-6.91 (m, 2H), 5.21-5.41 (m, 1H), 4.87 (d, 1H, J=16.0 Hz),4.51 (d, 1H, J=18.0 Hz), 4.39 (d, 1H, J=18.0 Hz), 4.20 (d, 1H, J=16.0Hz), 2.78-2.93 (m, 2H); EIMS: m/z: 493 (M+-HCl); HPLC purity: >95%

3,5-dibenzylidene-1-(S-2-amino-3-phenyl)-piperidin-4-one-HCl salt(RA181):¹H NMR (DMSO-d6): δ 7.82 (d, 2H, J=16.0 Hz), 7.17-7.75 (m, 15H),4.91-4.95 (m, 1H), 4.67 (d, 1H, J=16.0 Hz), 4.29-4.51 (m, 3H), 3.21-3.38(m, 2H); EIMS: m/z: 422 (M+-HCl); HPLC purity: >95%

3,5-bis(3,4-dichlorobenzylidene)-1-(S-2-amino-3-phenyl)-piperidin-4-one-HClsalt (RA190): ¹H NMR (DMSO-d6): δ 8.02 (s, 1H), 7.11-7.27 (m, 11H), 6.98(s, 2H), 4.95 (d, 1H, J=16.0 Hz), 4.68 (d, 1H, J=18.0 Hz), 4.51-4.68 (m,2H), 4.14 (d, 1H, J=18.0 Hz), 2.95 (dd, 1H, J=14.0 Hz), 2.80 (dd, 1H,J=14.0 Hz); 13C (DMSO-d6): 185.4, 174.9, 143.7, 141.5, 139.2, 138.3,135.6, 131.7, 129.9, 128.6, 127.1, 125.3, 54.7, 46.9, 42.1; EIMS: m/z:562 (M+-HCl); HPLC purity: 99% (21.7 min)

3,5-bis(3,4-dichlorobenzylidene)-1-(N-(1-oxo-S-3-phenyl-1-propan-2-yl)acetamide)-piperidin-4-one-HCl salt (RA190Ac): ¹H NMR (CDCl3): δ 8.12(s, 1H), 7.57 (s, 2H), 7.11-7.27 (m, 11H), 4.97 (dd, 1H, J=16.0 Hz),4.83 (d, 1H, J=18.0 Hz), 4.74 (d, 1H, J=18.0 Hz), 4.41 (d, 1H, J=16.0Hz), 4.12-4.19 (m, 1H), 3.19 (d, 1H, J=14.0 Hz), 3.08 (d, 1H, J=14.0Hz), 2.13 (s, 3H); EIMS: m/z: 603 (M+); Log P: 5.91, C Log P: 7.33; HPLCpurity: 99%

3,5-bis(3,4-dichlorobenzylidene)-1-(S-1H-imidazol-4-yl) piperidin-4-onedi HCl salt (RA196): ¹H NMR (DMSO-d6): δ 12.2 (s, 1H), 6.98-7.89 (m,10H), 4.96 (d, 1H, J=16.0 Hz), 4.74 (d, 1H, J=18.0 Hz), 4.39-4.41 (m,2H), 4.27 (d, 1H, J=18.0 Hz), 2.89-3.16 (m, 2H); EIMS: m/z: 551(M+-HCl); HPLC purity: >95%

3,5-bis(2-fluorobenzylidene)-1-(S-2-amino-3-phenyl)-piperidin-4-one-HClsalt (RA201): ¹H NMR (DMSO-d6): δ 7.96 (d, 2H, J=8.0 Hz), 7.21-7.58 (m,13H), 4.94-5.09 (m, 1H), 4.89 (d, 1H, J=16.0 Hz), 4.55 (d, 1H, J=18.0Hz), 4.43 (d, 1H, J=18.0 Hz), 4.260 (d, 1H, J=16.0 Hz), 2.76-2.97 (m,2H); EIMS: m/z: 458 (M+-HCl); HPLC purity: >95%.

3,5-bis(3,4-dichlorobenzylidene)-1-(S-2-amino-3-(4-hydroxy)-phenyl)-piperidin-4-oneHCl salt (RA213): ¹H NMR (DMSO-d6): δ 7.62 (s, 2H), 6.98-7.25 (m, 10H),4.99 (d, 1H, J=16.0 Hz), 4.75 (d, 1H, J=18.0 Hz), 4.59-4.72 (m, 2H),4.29 (d, 1H, J=18.0 Hz), 2.89-3.17 (m, 2H); EIMS: m/z: 474 (M+-HCl);HPLC purity: >95%

3,5-bis((2-hydroxyethylthio)(3,4-dichlorophenyl)methyl)-1-(S-2-amino-3-phenyl)-piperidin-4-one-HClsalt (RA190ME): ¹H NMR (CDCl3): 7.01-7.58 (m, 11H), 4.09-4.95 (m, 11H),3.17-3.98 (m, 4H), 2.23-2.87 (m, 4H); EIMS: m/z: 716 (M+-HCl); HPLCpurity: 95%.

HPLC conditions for RA190 purification.

TABLE 9 HPLC conditions for RA190 purification: % A (water/5% % Bacetonitrile, (Acetonitrile- Time (min) Flow (mL) 0.1% TFA) 0.1% TFA)0.01 1.00 80.0 20.0 5.00 6.00 80.0 20.0 20.00 6.00 5.0 95.0 30.00 6.0095.0 5.0 30.01 0.00 95.0 5.0

FIG. 17 shows a representative HPLC trace for RA190 purification, with apeak appearing after 21 minutes. High-performance liquid chromatography(HPLC), is a technique in analytic chemistry used to separate thecomponents in a mixture, to identify each component, and to quantifyeach component and to analyze the purity of given compound. Single peakat 21 minutes indicates RA190 eluting time is 21 minutes at given HPLCmethod and indicates the presence of single compound without impurities.

Additional Experiments

HeLa cells were treated with RA190, RA190B(RA190-Biotin) at variousconcentrations for the period of 12 h and cell lysate was subjected towestern blot analysis using anti Ubiquitin and PARP antibodies. Abuild-up of poly-ubiquitinated proteins in response to the inhibition ofthe proteasome by the treatments was observed. In addition, a westernblot analysis using anti-Ubiquitin and anti-PARP antibodies wasconducted and the RA190 treatments stimulated cleavage of PARP whichlead to cell death.

FIG. 18 shows the results of HeLa cells that were treated with RA190,RA190B (RA190-Biotin) and RA190R for the period of 48 h. Cell viabilitywas measured using MTT reagent. Significantly, the RA190 treatmentresulted in significant loss of cell viability.

20S Proteasome Chymotryptic Activity Assay:

FIG. 19 shows the results of a RA183, RA190 and RA195 20S proteasomeinhibition assay. All tested compounds were observed to inhibitproteasome activity. Purified 20S proteasome (500 ng) was incubated withdifferent RA compounds (1 μM) along with bortezomib (Bz, 1 uM) for 30min and 75 μM substrate (Suc-LLVY-AMC) was added and read the AMCrelease by measuring the fluorescence using fluorometer. 20S proteasomecleaves the chymotryptic substrate and releases fluorescent AMC. Whereasbortezomib inhibits this chymotryptic activity of the 20S proteasome,RA190, RA183, and RA195 do not at a concentration of 1 μM.

RA190 Inhibits TNFα-Induced NFκB Activation

293 cells transiently transfected with either NFκB/FL (fireflyluciferase reporter construct under control of an NFκB-driven promoter)or control CMV promoter-driven FL reporter construct were treated withcompounds and TNFα (10 ng/ml) for 7 h. Upon the addition of luciferin,bioluminescence was measured in cell lysates using a luminometer.Results can be seen in FIG. 20.

RA190 Effect on Pancreatic Cancer

FIGS. 21A-21F show a series of immunoblots and data graphs on the effectof various compounds on pancreatic cancer cells. 21A shows an immunoblotof PAO3C cell lysates. 21B shows an immunoblot showing the effect ofRA190 on the poly-ubiquitinated protein levels. 21C shows an immunoblotshowing the effect of RA190 on the levels of apoptotic proteins andactivated caspase-3. 21D shows an immunoblot showing the effect of RA190on CDK inhibitor p27. 21E shows a data graph showing the effect of RA190on Annexin V positive cells. 21F shows a data graph showing the effectof RA190 on active-caspase-3. FIG. 21B shows PAO3C cells treated withcompounds (1 μM) for the period of 12 h. Lysate was Subjected to westernblot and probed with Ubiquitin antibody. Actin used as referencecontrol, Bz=Bortezomib. Note: High molecular weight poly ubiquitinatedproteins in RA190 and RA195 lanes indicate inhibition of 19S proteasome.

FIG. 21B shows PAO3C cells treated with compounds (1 uM) for the periodof 12 h. Lysate was Subjected to western blot and probed with Ubiquitinantibody. Actin used as reference control, Bz=Bortezomib. Note: Highmolecular weight poly ubiquitinated proteins in RA190 and RA195 lanesindicate inhibition of 19S proteasome.

FIG. 21C shows PAO3C cells treated for the period of 24 h withcorresponding compounds (1 μM). Lysate subjected to western blot andprobed with Bax (A) and PARP (B) antibodies. Up regulation of bothproteins in the presence of compounds indicates apoptosis.

FIG. 21D shows PAO3C cells treated with compounds (1 μM) for the periodof 12 h. Lysate was subjected to western blot and probed with CDKinhibitor p27. Up regulation is seen with both compounds compared to thecontrol. Actin used as reference control. FIG. 21E shows PAO3C cellstreated with compounds for the period of 12 h and stained withPE-Annexin. Annexin V +ve cells were analyzed by FACS.

FIG. 21F shows BxPC-3 (left) and PAO3C (right) cells treated withcompounds for the period of 12 h and analyzed for active caspase-3positive cells by FACS.

Studies on RA195 and Analogs

Cell Viability Assay

Multiple Myeloma and isogenic Colon Cancer cells (HCT116) were treatedwith corresponding compounds for the period of 48 h and cell viabilitywas measured using XTT assay. IC50 values were determined in triplicateand are presented in μM. As seen in FIGS. 22A-D, compounds affect theviability of NCI-H929 (FIG. 22A), U266 (FIG. 22B) and HCT116 cells(FIGS. 22C and 22D). FIGS. 22A-22D show a series of data graphs showingthe effect of indicated compounds on the viability of various cancercell lines. 22A shows a data graph showing the effect of indicatedcompounds on NCI-H929 cell viability. 22B shows a data graph showing theeffect of indicated compounds on U266 cell viability. 22C and 22D showdata graphs showing the effect of indicated compounds on HCT116 cellviability.

RA195 Binds Covalently to Rpn13

Purified 19S proteasome (500 ng) was incubated with correspondingcompounds for 30 min and subjected to immunoblot and probed withHRP-conjugated streptavidin. In FIG. 23, the single band at 42 KDaindicates Rpn13 binding to 195. RA183B (20 μM) is used as positivecontrol for Rpn13 binding. Competition experiments were done with RA183(100 μM) and RA195 (100 μM) along with RA195B (20 μM). No binding wasobserved in the lanes of RA183 (20 μM) and RA195 (20 μM) alone and incompetition experiments.

RA195 Caused Accumulation of Poly Ubiquitinated Proteins and Elevationof Apoptotic Protein Bax in Pancreatic Cell Line

In FIG. 24, PAO3C cells were treated with RA195(195, 1 uM), Bortezomib(Bz, 1 μM) and DMSO (C) for the period of 12 h. Cell lysate wassubjected to immunoblot and probed for anti UB antibody. For Baxproteins levels cells were treated for 24 hr. Actin used as positivecontrol. The results show that RA195 causes accumulation of PolyUbiquitinated proteins (left) and elevation of apoptotic protein Bax(right).

RA195 and Analogs Stabilized UBFL In Vitro

HeLa cells were transiently transfected with tetra ubiquitin-fusedfirefly luciferase (4UB-FL) plasmid. After 48 h, the transfected cellswere treated with titrations of the indicated compounds for 4 h, andluciferase activity was measured. Data is expressed as a 4UB-FL foldchange compared to untreated cells. As seen in FIG. 25, the RA compoundsstabilize UBFL.

RA195 Induced Active Caspase-3 in HeLa Cells

HeLa cells were treated with RA195 (1 μM,) and Bz(1 μM) for the periodof 8 h. Cells were stained for PE-conjugated Caspase-3 antibody andmeasured active caspase-3 by FACS. As seen in FIG. 26, cells treatedwith RA195 (middle panel) have induced active capase expression comparedto those treated with Bortezomib (right panel) or control cells (leftpanel).

RA195 Induced Active Caspase-3 in Multiple Myeloma NCI-H929 Cells

NCI-H929 cells were treated with RA195 (0.5 μM) and Bz(0.5 μM) for theperiod of 8 h. Cells were stained for PE-conjugated Caspase-3 antibodyand measured active caspase-3 by FACS. As seen in FIG. 27, treatment ofNCI H929 cells with RA195, RA195AC, RA195BN and RA183 caused an increasein caspase-3 expression.

RA195 Induced Active Caspase-3 in Multiple Myeloma 0266 Cells

U266 cells were treated with RA195 (0.5 μM) and Bz(0.5 μM) for theperiod of 8 h. Cells were stained for PE-conjugated Caspase-3 antibodyand measured active caspase-3 by FACS. As seen in FIG. 28, treatment ofU266 cells with RA195, RA195AC, 195BN and RA183 caused an increase incaspase-3 expression.

RA195 and its Analog RA195Ac Inhibit TNFα-Induced NFκB Activation

293 cells transiently transfected with either NFκB/FL or control FLreporter genes were treated with compounds and TNFα (10 ng/ml) for 7 h.Upon the addition of luciferin, bioluminescence was measured in celllysates using a luminometer. As seen in FIG. 29, RA195 and its analogRA195Ac inhibited TNFα-induced NFκB activation.

RA195 Accumulated Poly UB Proteins and Elevated the Levels of p53 andp21 Proteins in HeLa Cells

HeLa cells were treated with corresponding compounds (1 μM) for 12 h andthe lysate was subjected to immunoblot and probed with anti-ubiquitin(UB), anti-p53, anti-p21 and anti-actin antibodies. As seen in FIG. 30,RA195 treatment caused the accumulation of poly-ubiqutinated (Poly UB)proteins and elevated the levels of p53 and p21 proteins in HeLa cells.

REFERENCES

-   1 Adams, J. (2004). The proteasome: a suitable antineoplastic    target. Nat Rev Cancer 4, 349-360.-   2 Al-Shami, A., Jhaver, K. G., Vogel, P., Wilkins, C., Humphries,    J., Davis, J. J., Xu, N., Potter, D. G., Gerhardt, B., Mullinax, R.,    et al. (2010). Regulators of the proteasome pathway, Uch37 and    Rpn13, play distinct roles in mouse development. PLoS ONE 5, e13654.-   3 Anchoori, R. K., Khan, S. R., Sueblinvong, T., Felthauser, A.,    lizuka, Y., Gavioli, R., Destro, F., Isaksson Vogel, R., Peng, S.,    Roden, R. B., and Bazzaro, M. (2011). Stressing the    ubiquitin-proteasome system without 20S proteolytic inhibition    selectively kills cervical cancer cells. PLoS ONE 6, e23888.-   4 Arastu-Kapur, S., Anderl, J. L., Kraus, M., Parlati, F., Shenk, K.    D., Lee, S. J., Muchamuel, T., Bennett, M. K., Driessen, C.,    Ball, A. J., et al. (2011). Nonproteasomal targets of the proteasome    inhibitors bortezomib and carfilzomib: a link to clinical adverse    events. Clin. Cancer Res. 17, 2734-2743.-   5 Bazzaro, M., Anchoori, R. K., Mudiam, M. K., Issaenko, O., Kumar,    S., Karanam, B., Lin, Z., Isaksson Vogel, R., Gavioli, R., Destro,    F., et al. (2011). a,b-Unsaturated carbonyl system of chalcone-based    derivatives is responsible for broad inhibition of proteasomal    activity and preferential killing of human papilloma virus (HPV)    positive cervical cancer cells. J. Med. Chem. 54, 449-456.-   6 Bazzaro, M., Lee, M. K., Zoso, A., Stirling, W. L., Santillan, A.,    Shih le, M., and Roden, R. B. (2006). Ubiquitin-proteasome system    stress sensitizes ovarian cancer to proteasome inhibitor-induced    apoptosis. Cancer Res 66, 3754-3763.-   7 Bedford, L., Lowe, J., Dick, L. R., Mayer, R. J., and    Brownell, J. E. (2011). Ubiquitin-like protein conjugation and the    ubiquitin-proteasome system as drug targets. Nat Rev Drug Discov 10,    29-46.-   8 Best, S. R., Peng, S., Juang, C. M., Hung, C. F., Hannaman, D.,    Saunders, J. R., Wu, T. C., and Pai, S. I. (2009). Administration of    HPV DNA vaccine via electroporation elicits the strongest CD8+ T    cell immune responses compared to intramuscular injection and    intradermal gene gun delivery. Vaccine 27, 5450-5459.-   9 Chauhan, D., Catley, L., Li, G., Podar, K., Hideshima, T.,    Velankar, M., Mitsiades, C., Mitsiades, N., Yasui, H., Letai, A., et    al. (2005). A novel orally active proteasome inhibitor induces    apoptosis in multiple myeloma cells with mechanisms distinct from    Bortezomib. Cancer Cell 8, 407-419.-   10 Chen, S., Blank, J. L., Peters, T., Liu, X. J., Rappoli, D. M.,    Pickard, M. D., Menon, S., Yu, J., Driscoll, D. L., Lingaraj, T., et    al. (2010a). Genome-wide siRNA screen for modulators of cell death    induced by proteasome inhibitor bortezomib. Cancer Res 70,    4318-4326.-   11 Chen, X., Lee, B. H., Finley, D., and Walters, K. J. (2010b).    Structure of proteasome ubiquitin receptor hRpn13 and its activation    by the scaffolding protein hRpn2. Mol Cell 38, 404-415.-   12 Ciechanover, A. (1998). The ubiquitin-proteasome pathway: on    protein death and cell life. Embo J 17, 7151-7160.-   13 Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J.,    and Bax, A. (1995). NMRPipe: a multidimensional spectral processing    system based on UNIX pipes. J Biomol NMR 6, 277-293.-   14 Deveraux, Q., Ustrell, V., Pickart, C., and Rechsteiner, M.    (1994). A 26 S protease subunit that binds ubiquitin conjugates. J.    Biol. Chem. 269, 7059-7061.-   15 Dominguez, C., Boelens, R., and Bonvin, A. M. (2003). HADDOCK: a    protein-protein docking approach based on biochemical or biophysical    information. J Am Chem Soc 125, 1731-1737.-   16 Gandhi, T. K., Zhong, J., Mathivanan, S., Karthick, L.,    Chandrika, K. N., Mohan, S. S., Sharma, S., Pinkert, S., Nagaraju,    S., Periaswamy, B., et al. (2006). Analysis of the human protein    interactome and comparison with yeast,-   worm and fly interaction datasets. Nat. Genet. 38, 285-293.-   17 Hamazaki, J., lemura, S., Natsume, T., Yashiroda, H., Tanaka, K.,    and Murata, S. (2006). A novel proteasome interacting protein    recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. Embo    J 25, 4524-4536.-   18 Howie, H. L., Katzenellenbogen, R. A., and Galloway, D. A.    (2009). Papillomavirus E6 proteins. Virology 384, 324-334.-   19 Husnjak, K., Elsasser, S., Zhang, N., Chen, X., Randles, L., Shi,    Y., Hofmann, K., Walters, K. J., Finley, D., and Dikic, I. (2008).    Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature 453,    481-488.-   20 Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., and    Sakaki, Y. (2001). A comprehensive two-hybrid analysis to explore    the yeast protein interactome. Proc. Natl. Acad. Sci. USA 98,    4569-4574.-   21 Koulich, E., Li, X., and DeMartino, G. N. (2008). Relative    structural and functional roles of multiple deubiquitylating    proteins associated with mammalian 26S proteasome. Mol. Biol. Cell    19, 1072-1082.-   22 Kuhn, D. J., Berkova, Z., Jones, R. J., Woessner, R.,    Bjorklund, C. C., Ma, W., Davis, R. E., Lin, P., Wang, H.,    Madden, T. L., et al. (2012). Targeting the insulin-like growth    factor-1 receptor to overcome bortezomib resistance in preclinical    models of multiple myeloma. Blood 120, 3260-3270.-   23 Lin, Z., Bazzaro, M., Wang, M. C., Chan, K. C., Peng, S., and    Roden, R. B. (2009). Combination of Proteasome and HDAC Inhibitors    for Uterine Cervical Cancer Treatment. Clin Cancer Res 15, 570-577.-   24 Luker, G. D., Pica, C. M., Song, J., Luker, K. E., and    Piwnica-Worms, D. (2003). Imaging 26S proteasome activity and    inhibition in living mice. Nat Med 9, 969-973.-   25 Maki, C. G., Huibregtse, J. M., and Howley, P. M. (1996). In vivo    ubiquitination and proteasome-mediated degradation of p53(1). Cancer    Res 56, 2649-2654.-   26 Moody, C. A., and Laimins, L. A. (2010). Human papillomavirus    oncoproteins: pathways to transformation. Nat Rev Cancer 10,    550-560.-   27 Qiu, X. B., Ouyang, S. Y., Li, C. J., Miao, S., Wang, L., and    Goldberg, A. L. (2006). hRpn13/ADRM1/GP110 is a novel proteasome    subunit that binds the deubiquitinating enzyme, UCH37. EMBO J. 25,    5742-5753.-   28 Ri, M., Iida, S., Nakashima, T., Miyazaki, H., Mori, F., Ito, A.,    Inagaki, A., Kusumoto, S., Ishida, T., Komatsu, H., et al. (2010).    Bortezomib-resistant myeloma cell lines: a role for mutated PSMBS in    preventing the accumulation of unfolded proteins and fatal ER    stress. Leukemia 24, 1506-1512.-   29 Ruschak, A. M., Slassi, M., Kay, L. E., and Schimmer, A. D.    (2011). Novel proteasome inhibitors to overcome bortezomib    resistance. J Natl Cancer Inst 103, 1007-1017.-   30 Sakata, E., Bohn, S., Mihalache, O., Kiss, P., Beck, F., Nagy,    I., Nickell, S., Tanaka, K., Saeki, Y., Forster, F., et al. (2012).    Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13    by electron cryomicroscopy. Proc Natl Acad Sci USA 109, 1479-1484.-   31 Schreiner, P., Chen, X., Husnjak, K., Randles, L., Zhang, N.,    Elsasser, S., Finley, D., Dikic, I., Walters, K. J., and Groll, M.    (2008). Ubiquitin docking at the proteasome through a novel    pleckstrin-homology domain interaction. Nature 453, 548-552.-   32 Schwartz, A. L., and Ciechanover, A. (2009). Targeting proteins    for destruction by the ubiquitin system: implications for human    pathobiology. Annu Rev Pharmacol Toxicol 49, 73-96.-   33 Spisek, R., and Dhodapkar, M. V. (2007). Towards a better way to    die with chemotherapy: role of heat shock protein exposure on dying    tumor cells. Cell Cycle 6, 1962-1965.-   34 Trimble, C., Lin, C. T., Hung, C. F., Pai, S., Juang, J., He, L.,    Gillison, M., Pardoll, D., Wu, L., and Wu, T. C. (2003). Comparison    of the CD8+ T cell responses and antitumor effects generated by DNA    vaccine administered through gene gun, biojector, and syringe.    Vaccine 21, 4036-4042.-   35 Vousden, K. H., and Lu, X. (2002). Live or let die: the cell's    response to p53. Nat Rev Cancer 2, 594-604.-   36 Welters, M. J., Kenter, G. G., Piersma, S. J., Vloon, A. P.,    Lowik, M. J., Berends-van der Meer, D. M., Drijfhout, J. W.,    Valentijn, A. R., Wafelman, A. R., Oostendorp, J., et al. (2008).    Induction of tumor-specific CD4+ and CD8+ T-cell immunity in    cervical cancer patients by a human papillomavirus type 16 E6 and E7    long peptides vaccine. Clin Cancer Res 14, 178-187.-   37 Yao, T., Song, L., Xu, W., DeMartino, G. N., Florens, L.,    Swanson, S. K., Washburn, M. P., Conaway, R. C., Conaway, J. W., and    Cohen, R. E. (2006) Proteosome Recruitment and activation of the    Uch37 debiquitinating enzyme by Adrm1. Nat. Cell Biol. 8, 994-1002

The following claims are thus to be understood to include what isspecifically illustrated and described above, what is conceptuallyequivalent, what can be obviously substituted and also what essentiallyincorporates the essential idea of the invention. Those skilled in theart will appreciate that various adaptations and modifications of thejust-described preferred embodiment can be configured without departingfrom the scope of the invention. The illustrated embodiment has been setforth only for the purposes of example and that should not be taken aslimiting the invention. Therefore, it is to be understood that, withinthe scope of the appended claims, the invention may be practiced otherthan as specifically described herein.

What is claimed is:
 1. A method of inhibiting proteasomes in a mammal byadministering to the mammal an effective amount of a compound of formulaI,

wherein each pair of As is one of: (i) phenyl, optionally substitutedwith 1-5 substituents selected from the group consisting of R1, OR1,NR1R2, S(O)_(q)R1, SO₂NR1R2, NR1SO₂R2, C(O)R1, C(O)OR1, C(O)NR1R2,NR1C(O)R2, NR1C(O)OR2, CF₃, and OCF₃, (ii) naphthyl, optionallysubstituted with 1-5 substituents selected from the consisting of R1,OR1, NR1R2, S(O)_(q)R1, SO₂NR1R2, NR1SO₂R2, C(O)R1, C(O)OR1, C(O)NR1R2,NR1C(O)R2, NR1C(O)OR2, CF₃, and OCF₃, (iii) a 5 or 6 membered monocyclicheteroaryl group, having 1-3 heteroatoms selected from the groupconsisting of 0, N, and S, optionally substituted with 1-3 substituentsselected from the group consisting of R1, OR1, NR1R2, S(O)_(q)R1,SO₂NR1R2, NR1SO₂R2, C(O)R1, C(O)OR1, C(O)NR1R2, NR1C(O)R2, NR1C(O)OR2,CF₃, and OCF₃, and (iv) an 8 to 10 membered bicyclic heteroaaryl groupcontaining 1-3 heteroatoms selected from the group consisting of O, N,and S; and the second ring is fused to the first ring using 3 to 4carbon atoms, and the bicyclic hetero aryl group is optionallysubstituted with 1-3 substituents selected from the group consisting ofR1, OR1, NR1R2, S(O)_(q)R1, SO₂NR1R2, NR1SO₂R2, C(O)R1, C(O)OR1,C(O)NR1R2, NR1 C(O)R2, NR1C(O)OR2, CF₃, and OCF₃; wherein X is OR1 orNP, wherein P is selected from the group consisting of R1, C(O)R1,C(O)OR1, C(O)NR1R2, S—N(R1)COOR1, and S—N(R1), wherein Y is selectedfrom the group consisting of O, S, NR1 and CR1R2, wherein R1 and R2 areselected from the group consisting of hydrogen, nitro, hydroxyl,carboxy, amino, halogen, cyano and C₁-C₁₄ linear or branched alkylgroups, that are optionally substituted with 1-3 substituents selectedfrom the group consisting of C₁-C₁₄ linear or branched alkyl, up toperhalo substituted C₁-C₁₄ linear or branched alkyl, C₁-C₁₄ alkoxy,hydrogen, nitro, hydroxyl, carboxy, amino, C₁-C₁₄ alkylamino, C₁-C₁₄dialkylamino, halogen, and cyano; and wherein Z is selected from thegroup consisting of hydrogen; C₁ to C₁₄ linear, branched, or cyclicalkyls; phenyl; benzyl, 1-5 substituted benzyl, C₁ to C₃ alkyl-phenyl,wherein the alkyl moiety is optionally substituted with halogen up toperhalo; up to perhalo substituted C₁ to C₁₄ linear or branched alkyls;—(CH₂)_(q)—K, where K is a 5 or 6 membered monocyclic heterocyclic ring,containing 1 to 4 atoms selected from oxygen, nitrogen and sulfur, whichis saturated, partially saturated, or aromatic, or an 8 to 10 memberedbicyclic heteroaryl having 1-4 heteroatoms selected from the groupconsisting of O, N and S, wherein said alkyl moiety is optionallysubstituted with halogen up to perhalo; and wherein the variable q is aninteger ranging from 0 to 4; and wherein inhibiting the proteasomescomprises inhibiting a ubiquitin proteasome system.
 2. The method ofclaim 1, wherein the mammal is a human.
 3. The method of claim 1,wherein the method treats a condition or a disease in the mammal.
 4. Themethod of claim 3, wherein the mammal is a human.
 5. The method of claim3, wherein the condition or disease is a type of cancer.
 6. The methodof claim 5, wherein the type of cancer is selected from the groupconsisting of breast cancer, cervical cancer, ovarian cancer, multiplemyeloma, breast cancer and pancreatic cancer.
 7. The method of claim 5,wherein the type of cancer is associated with Human Papilloma Virus(HPV).
 8. The method of claim 1, wherein the compound is administered incombination with at least one other therapeutic agent.
 9. The method ofclaim 8, wherein the at least one other therapeutic agent is aproteasome inhibitor.
 10. The method of claim 8, wherein the at leastone other therapeutic agent is bortezomib.
 11. The method of claim 1,wherein the compound binds to RPN13.
 12. The method of claim 1, whereinthe compound is orally administered.
 13. The method of claim 1, whereinthe compound is intraperitoneally administered.
 14. The method compoundof claim 1, wherein the compound is topically applied.
 15. The method ofclaim 1, wherein the compound has the structure:


16. The method of claim 1, wherein the compound has the structure:


17. The method of claim 1, wherein the compound has the structure: